Method of detecting coccidioides species

ABSTRACT

The present technology provides methods and kits that may be used to detect and quantify the presence of  Coccidioides  species. The methods include quantification real-time PCR assays, and the kits and compositions include oligonucleotides used as primers and probes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation-In-Part of U.S. patent applicationSer. No. 13/935,668, filed Jul. 5, 2013 (published as US 20140011693),which claims the benefit of U.S. Provisional Application No. 61/668,203,filed Jul. 5, 2012; this application also claims the benefit of U.S.Provisional Application No. 62/319,612, filed Apr. 7, 2016. The entirecontents and disclosure of the above-mentioned applications are hereinincorporated by reference in their entireties.

INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY FILED

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 8 kilobyte ASCII (text) file named“Seq_list” created on Jul. 15, 2016.

FIELD OF THE TECHNOLOGY

The present technology provides methods and kits for specificallydetecting and quantifying Coccidioides in a sample, including anenvironmentally derived sample, such as soil.

BACKGROUND OF THE TECHNOLOGY

Coccidioidomycosis is caused by infection with Coccidioides immitis orCoccidioides posadasii (collectively “Coccidioides”). C. immitis and C.posadasii are the fungal etiologic agents of coccidioidomycosis (a.k.a.Valley Fever) and are endemic to arid soils of the southwest UnitedStates, as well as parts of Mexico, and Central and South America.Primary hosts acquire Coccidioides via inhalation of aerosolizedarthroconidia upon soil disruption. Coccidioidomycosis most commonlycauses a progressive pulmonary infection in humans and other vertebratehosts but also can disseminate to other body parts including the skin,brain, bone, and meninges. This disseminated secondarycoccidioidomycosis often is severe and can result in patient death.However, in cases where infection is resolved, patients usually acquirea specific and lifelong immunity to the fungus.

Coccidioidomycosis infection rates have increased dramatically in thelast decade with the State of Arizona documenting the number of reportedcases per 100,000 people having increased from 20.8 in 1997 to 186.0 in2010. Increased physician awareness and testing likely accounts for aportion of this case increase. An additional cause for this increase maybe influxes of immunologically naïve individuals into Arizona. Asignificant number of individuals from outside the Coccidioides endemicregion migrate annually to the desert southwest and are at greater riskfor developing coccidioidomycosis, even after returning to theirrespective homes. These infections, therefore, are likely to escape orconfound diagnosis in non-endemic regions.

While Real-Time PCR based assays have been developed that can helpclinicians identify Coccidioides as a cause of illness, these assayshave lacked needed sensitivity and do not accurately quantify the loadof Coccidioides organisms in an infection.

BRIEF SUMMARY OF THE TECHNOLOGY

Provided herein is a method of determining the presence or absence ofCoccidioides in a DNA-containing sample. The general method comprisesthe steps of: (1) adding a first and a second oligonucleotide capable ofbinding SEQ ID NO. 1 to a mixture comprising the DNA-containing sample,wherein the first oligonucleotide includes at least one sequenceselected from the group consisting of SEQ ID NOs: 10-20 andoligonucleotides having at least 90% sequence identity to any one of SEQID NOs: 10-20, wherein the second oligonucleotide includes at least onesequence selected from the group comprising SEQ ID NOs: 21-38, andoligonucleotides having at least 90% sequence identity to any one of SEQID NOs: 21-38. The method may also include (2) subjecting the mixturecontaining the first and second oligonucleotides to conditions thatallow amplification of nucleic acid comprising the firstoligonucleotide, (3) obtaining a result indicating nucleic acidamplification comprising the first oligonucleotide; and (4) determiningthe presence or absence of Coccidioides in the DNA-containing samplebased on the result. In some examples, the result obtained by thegeneral method comprises a Ct value. The general method as set forthabove may further comprise the step of adding a third oligonucleotide tothe mixture, wherein the third oligonucleotide binds to its complementincluded in the amplification products by the first and secondoligonucleotides. The third oligonucleotide preferably includes asequence selected from the group consisting of SEQ ID NO. 2 and homologsthereof having at least 90% sequence identity and complementarity undersimilar stringency. In the general method, at least one of the first,the second and the third oligonucleotides comprises a label. For someexamples, the label may comprise a fluorescent label selected from thegroup consisting of FAM, dR110, 5-FAM, 6FAM, dR6G, JOE, HEX, VIC, TET,dTAMRA, TAMRA, NED, dROX, PET, BHQ+, Gold540, MGB-NFQ, and LIZ. In oneexample, the third oligonucleotide comprises a fluorescent labelselected from the group consisting of FAM, dR110, 5-FAM, 6FAM, dR6G,JOE, HEX, VIC, TET, dTAMRA, TAMRA, NED, dROX, PET, BHQ+, Gold540,MGB-NFQ, and LIZ. In some preferred embodiments, when at least one, twoor all of the first, second, and third oligonucleotides include labels,the labels are preferably different for the at least two and preferablyfor all of the first, second, and third oligonucleotides, respectively.

The general method as provided may further comprise the step ofisolating DNA from the DNA-containing sample. The sample may comprise anenvironmental sample or may be derived from a subject. In preferredforms, the subject is selected from the group consisting of a human, acompanion animal, a livestock animal, and a wild animal species. Inother preferred aspects, the environmental sample may be a soil sample.

Also provided is a method of quantifying Coccidioides in aDNA-containing sample. The method may comprise the steps of: (1) addinga first and a second oligonucleotide capable of binding SEQ ID NO. 1 toa mixture comprising the DNA-containing sample, wherein the firstoligonucleotide includes at least one sequence selected from the groupconsisting of SEQ ID NOs: 10-20 and oligonucleotides having at least 90%sequence identity to any one of SEQ ID NOs: 10-20, wherein the secondoligonucleotide includes at least one sequence selected from the groupcomprising SEQ ID NOs: 21-38, and oligonucleotides having at least 90%sequence identity to any one of SEQ ID NOs: 21-38; (2) subjecting themixture containing the first and second oligonucleotides to conditionsthat allow amplification of a template DNA comprising the firstoligonucleotide; (3) obtaining a first result indicating amplificationof the template DNA and Coccidioides quantification; and (4) calculatingCoccidioides quantification based on the first result in comparison to areference result, wherein Coccidioides quantification determines theamount of template DNA in the sample. In some example, the referenceresult is obtained by the same quantification method using aDNA-containing sample having a known quantity of Coccidioides. In someother example, the reference result is predetermined. Sometimes, each ofthe first and the reference result comprises a Ct value.

The quantification method may further comprise the step of adding athird oligonucleotide to the mixture, wherein the third oligonucleotidebinds to its complement included in the amplification products by thefirst and second oligonucleotides. In one example, the thirdoligonucleotide includes a sequence selected from the group consistingof SEQ ID NO. 2 and homologs thereof having at least 90% sequenceidentity and complementarity under similar stringency. In thequantification method, at least one of the first and the secondoligonucleotides comprises a label. In some preferred forms, if morethan one of the first, second, or third oligonucleotides are used andmore than one of these includes a label, the labels will be differentfor the first, second, and third nucleotides, respectively. In someexamples, the label comprises a fluorescent label selected from thegroup consisting of FAM, dR110, 5-FAM, 6FAM, dR6G, JOE, HEX, VIC, TET,dTAMRA, TAMRA, NED, dROX, PET, BHQ+, Gold540, MGB-NFQ, and LIZ. In oneexample, the third oligonucleotide comprises a fluorescent labelselected from the group consisting of FAM, dR110, 5-FAM, 6FAM, dR6G,JOE, HEX, VIC, TET, dTAMRA, TAMRA, NED, dROX, PET, BHQ+, Gold540,MGB-NFQ, and LIZ.

The quantification method may further comprise the step of isolating DNAfrom the DNA-containing sample. In some examples, the sample comprisesan environmental sample. In other examples, the sample is derived from asubject, preferably the subject is selected from the group consisting ofa human, a companion animal, and a livestock animal. In some additionalembodiments, the environmental sample may comprise a soil sample.

Other aspects and iterations of the technology are described in moredetail below.

DETAILED DESCRIPTION OF THE TECHNOLOGY

The present technology discloses assays, methods and kits designed todetect and quantify total Coccidioides sp in a sample. This technologyprovides a genomic target specific to Coccidioides sp, including C.immitis and C. posadasii, and other Coccidiodies species. A real-timequantitative Polymerase Chain Reaction (real-time qPCR) based assay,providing a straightforward, highly sensitive and specific assay systemfor rapidly detecting and quantifying Coccidioides in a sample, isprovided based on the genomic target disclosed herein.

I. Species or Strain Specific Sequences

Species or strain specific sequences are sequences unique to the speciesor strain, that is, not shared by other previously characterized speciesor strains. The species specific sequences identified in C. immitis andC. posadasii often differ only by a single nucleotide, which is calledSNP (single nucleotide polymorphism). The strain specific SNP, is alsocalled allelic identification herein, signifies the identity of C.immitis or C. posadasii. The concept of “allele” or “allelic” isdetailed below.

When a particular species or strain specific sequence is identified,probes or primers may be designed based on any part of that sequence.The probes or primers may also be the entirety of that sequence. Theprimers or probes designed according to a particular species or strainsequence, or alleles thereof, may also be represented in degenerateform, or comprise chemically modified nucleic acids, or any othercomponents that facilitate the identification of the identifyingsequence of a strain or species. The concept of a sequence identified tobe specific to a species or strain further encompasses nucleic acidsequences that are less than 100% identical to the specific sequence,but are still capable of specifically detecting the species or strain.Note that in a nucleic acid sequence, T or U may be used interchangeablydepending on whether the nucleic acid is DNA or RNA. A sequence havingless than 60% 70%, 80%, 90%, 95%, 99% or 100% identity to theidentifying sequence or allele thereof may still be encompassed by thetechnology if it is capable of binding to its complementary sequenceand/or facilitating nucleic acid amplification of a desired targetsequence.

As used herein, the term “sample” may refer to any source in whichCoccidioides nucleic acids may be detectable. A sample may be derivedfrom anywhere that fungus or any part of a fungal body may be foundincluding soil, air, water, solid surfaces (whether natural orartificial) culture media, foodstuffs, and any interfaces between orcombinations of these elements. Additionally, a sample may be derivedfrom a subject, such as a plant or animal, including humans. Samplesderived from animals include but are not limited to biopsy or other invivo or ex vivo analysis of prostate, breast, skin, muscle, facia,brain, endometrium, lung, head and neck, pancreas, small intestine,blood, liver, testes, ovaries, colon, skin, stomach, esophagus, spleen,lymph node, bone marrow, kidney, placenta, or fetus. Samples derivedfrom subjects may also take the form of a fluid sample such asperipheral blood, lymph fluid, ascites, serous fluid, pleural effusion,sputum, bronchial wash, bronchioalveolar lavage fluid (BALF),cerebrospinal fluid, semen, amniotic fluid, lacrimal fluid, stool,urine, hair, or any other source in which a fungus, or any part of afungus might be present. Samples collected from a plant may be collectedfrom part of a plant or from an entire plant. Samples may be collectedby any method now known or yet to be disclosed, including swiping orswabbing an area or orifice, removal of a piece of tissue as in abiopsy, or any method known to collect bodily fluids. Samples may alsoinclude Coccidioides that has been previously isolated from one or moreprior samples and grown in an isolated environment (e.g., a laboratory).Thereafter, one or more biomolecules (e.g., nucleic acids or protein)can be isolated from the Coccidioides for used in the methods disclosedherein.

An allele includes any form of a particular nucleic acid that may berecognized as a form of existence of a particular nucleic acid onaccount of its location, sequence, modification, or any othercharacteristics that may identify it as being a particular existing formof that particular nucleic acid. Alleles include, but need not belimited to, forms of a nucleic acid that include point mutations,deletions, single nucleotide polymorphisms (SNPs), inversions,translocations, heterochromatic insertions, and differentiallymethylated sequences relative to a reference gene, whether alone or incombination. When a particular nucleic acid is a gene, the allele ofthis particular gene may or may not produce a functional protein; thefunctional protein thereof may or may not comprise a silent mutation, orframe-shift mutation. The different alleles of a particular gene mayeach produce a protein with altered function, localization, stability,dimerization, or protein-protein interaction; and may haveoverexpression, under-expression or no expression; may have alteredtemporal or spatial expression specificity. The presence or absence ofan allele may be detected through the use of any process known in theart, including using primers and probes designed accordingly for PCR,sequencing, hybridization analyses. An allele may also be called amutation or a mutant. An allele may be compared to another allele thatmay be termed a wild type form of an allele. In some cases, the wildtype allele is more common than the mutant.

The term “primer” refers to an oligonucleotide, whether occurringnaturally as in a purified restriction digest or produced synthetically,which is capable of acting as a point of initiation of synthesis whenplaced under conditions in which synthesis of a primer extension productwhich is complementary to a nucleic acid strand is induced, i.e., in thepresence of nucleotides and an inducing agent such as DNA polymerase andat a suitable temperature and pH. The primer is preferablysingle-stranded for maximum efficiency in amplification. Alternatively,the primer is first treated to ensure that it is single-stranded beforebeing used to prepare extension products. Preferably, the primer is anoligodeoxyribonucleotide. The primer must be sufficiently long to primethe synthesis of extension products in the presence of the inducingagent. The exact lengths of the primers will depend on many factors,including temperature, source of primer and the use of the method.Oligonucleotides, such as a probe or primer, containing a sequencecomplementary to a sequence specific to a Coccidioides species or strainwill typically not hybridize to the corresponding portion of the genomeof other species or strains under stringent conditions. Understood bythose skilled in the art, for example, high stringent hybridizationconditions are equivalent to: 5×SSPE, 0.5% SDS, 5×Denhardt's reagent and100 μg/ml denatured salmon sperm DNA at 42° C. followed by washing in asolution comprising 0.1×SSPE, 1.0% SDS at 42° C. when a probe of about500 nucleotides in length is employed, and washed with 2×SSC, 0.1% SDSfollowed by 0.1×SSC, 0.1% SDS. Stringent conditions in PCR reaction maybe controlled by temperature or by the concentration of certain salt inthe buffer.

Primers and probes that are designed based on strain specific genes,allelic discriminative nucleic acid, or alleles thereof, are often usedto screen samples to specifically and selectively detect the presence orabsence of a particular species or strain of a bacteria, fungus, virus,or a pathogen thereof. The detection using primers and probes may bethrough various methods including PCR-based (polymerase chainreaction-based) methods such as real-time PCR, quantitative PCR,quantitative real time PCR; allele specific ligation; comparativegenomic hybridization; sequencing; and other methods known in the art.One aspect of the present technology provides primers based onCoccidioides specific sequence for quantitative PCR assays comprisingone or more specific primer sets and probes to detect the presence ofCoccidioides DNA.

When a nucleic acid includes a particular sequence, the sequence may bea part of a longer nucleic acid or may be the entirety of the sequence.The nucleic acid may contain nucleotides 5′ of the sequence, 3′ of thesequence, or both. The concept of a nucleic acid including a particularsequence further encompasses nucleic acids that contain less than thefull sequence that are still capable of specifically detecting a marker.Nucleic acid sequences may be identified by the IUAPC letter code whichis as follows: A—Adenine base; C—Cytosine base; G—guanine base; T orU—thymine or uracil base. For some degenerate primers oroligonucleotides, the following abbreviations may be used to providesequence information: M-A or C; R-A or G; W-A or T; S-C or G; Y-C or T;K-G or T; V-A or C or G; H-A or C or T; D-A or G or T; B-C or G or T; Nor X-A or C or G or T. Note that T or U may be used interchangeablydepending on whether the nucleic acid is DNA or RNA.

As to probes, they may be used for single probe analysis or multiplexprobe/primer combined Real Time PCR and quantitative PCR (qPCR)analysis. Oligonucleotide probes complementary to a selected sequencewithin the target sequence defined by the amplification region by theprimers may be designed. In one exemplary example, oligonucleotideprobes facilitating Real Time-PCR/qPCR product detection arecomplementary to a selected sequence within the target sequencedownstream from either the upstream or downstream primer. Therefore,these probes hybridize to an internal sequence of the amplified fragmentof a targeted sequence.

Many assays detecting the presence of a target can also quantify theamount of the target in a given sample. In particular, when there isonly one copy of the identified strain specific genes, alleles thereof,or other allelic discriminative nucleic acid in a fungal genome, theprimers and probes designed to specifically and selectively detect thepresence or absence of such single copy target may be further used toquantify the amount of Coccidioides spp in a sample. In one embodiment,the Coccidioides quantitative diagnosis assay (“CocciDxQ” hereafter) asprovided herein is used to quantify Coccidioides via a region that isassociated with copia-like retrotransposon family protein found inCoccidioides posadasii C735 delta SOWgp (GenBank Accession XM003069703.1; SEQ IDNO:1—TGTTAGGTAATCCAACTAGCACCTCGCTCACGTGACCCACATAGATTAGCCGAGATTCCCCTTTAGGTAGCTTAGTGAATGACAAGCATACAAGTCCTCCATCA) specific toCoccidioides species.

In some embodiments, the copia-like retrotransposon family protein foundin Coccidioides posadasii C735 delta SOWgp (SEQ ID NO: 1) can beemployed with additional assays. For example, some embodiments providean assay that can be used to detect Coccidioides in samples, such as asoil sample or a sample derived from a non-environmental source(hereinafter “CocciENV”). For example, CocciENV can be used to detectthe presence of one or more species of Coccidioides in a soil sample forany downstream purposes, such as establishing an area of endemic ValleyFever. In other embodiments, CocciENV can be used for diagnostic or anyother purposes desired by the user.

In some embodiments, the CocciDxQ assay is a real-time PCR that employsa probe and a multiplex set of forward primers and reverse primers thattarget part or all of the target sequence represented by SEQ ID NO: 1.In one embodiment, the probe is labeled with fluorescence. In anotherembodiment, the probe comprises a 6FAM and an MGB-NFQ label. In oneembodiment the probe comprises a sequence represented by SEQ ID NO: 2 orhomologs of SEQ ID NO: 2 with at least 80%, more preferably 90%, stillmore preferably 91%, even more preferably 92%, still more preferably93%, even more preferably 94%, still more preferably 95%, even morepreferably 96%, still more preferably 97%, even more preferably 98%,still more preferably 99%, and most preferably 99.8% or more identityand complementarity under similar stringency. In one embodiment, theCocciDxQ assay as disclosed herein comprises at least one forward primerand at least one reverse primer comprising primer sequences representedby SEQ ID NOs in Table 1 or homologs of SEQ ID NOs in Table 1 with atleast 80% more preferably 90%, still more preferably 91%, even morepreferably 92%, still more preferably 93%, even more preferably 94%,still more preferably 95%, even more preferably 96%, still morepreferably 97%, even more preferably 98%, still more preferably 99%, andmost preferably 99.8% or more identity and complementarity under similarstringency. In one embodiment, the forward primers comprise one or moredegenerative primers. In another embodiment, the reverse primerscomprise one or more degenerative primers. In yet another embodiment,both the forward primers and the reverse primers comprise one or moredegenerative primers. In some embodiments, the CocciDxQ assay maycomprise more than 1 forward primer and more than 1 reverse primer. Forexample, the CocciDxQ assay may comprise two, three, four and moreprimers; as such, the CocciDxQ assay may comprise two forward primersand one reverse primer, or two forward primers and two reverse primers,or three forward primers and one reverse primer. In one embodiment, theCocciDxQ assay comprises three forward primers and four reverse primersrepresented by SEQ ID NOs: 3-9 (Table 1).

TABLE 1 CocciDxQ Assay Probe Name Probe Sequence 5′ to 3′ SEQ ID NOCQ_3_probe ACCCACATAGATTAGC SEQ ID NO: 2 Forward Primer Name ForwardPrimer Sequence 5′ to 3′ CQ_3_F_v2a GTGTTAGGTAGTCCAACTAGCACCT SEQ ID NO:3 CQ_3_F_v2b GTGTTAGGTAATCCAACCAGCACCT SEQ ID NO: 4 CQ_3_F_v2cGTGTTAGGTAATCCAACTAGCACCT SEQ ID NO: 5 Reverse Primer Name ReversePrimer Sequence 5′ to 3′ CQ_3_R_v2a CTGATGGAGGACTCGTATGCTTGT SEQ ID NO:6 CQ_3_R_v2b CTGATGGAGGACTTGTACACTTGT SEQ ID NO: 7 CQ_3_R_v2cCTGATGGAGGAATTGTATGCTTGT SEQ ID NO: 8 CQ_3_R_v2dCTGATGGAGGACTTGTATGCTTGT SEQ ID NO: 9

The provided assays can detect less than one genomic DNA molecule permicroliter of DNA, which sensitivity is imparted by high genomic copynumber of the target gene, 85 copies/genome.

In some embodiments, the CocciENV assay is a real-time PCR that employsa probe and a multiplex set of forward primers and reverse primers thattarget part or all of the target sequence represented by SEQ ID NO: 1.In one embodiment, the probe is labeled with fluorescence. In anotherembodiment, the probe comprises a 6FAM and an MGB-NFQ label. In oneembodiment the probe comprises a sequence represented by SEQ ID NO: 2 orhomologs of SEQ ID NO: 2 with at least 80%, more preferably 90%, stillmore preferably 91%, even more preferably 92%, still more preferably93%, even more preferably 94%, still more preferably 95%, even morepreferably 96%, still more preferably 97%, even more preferably 98%,still more preferably 99%, and most preferably 99.8% or more identityand complementarity under similar stringency. In one embodiment, theCocciENV assay as disclosed herein comprises at least one forward primerand at least one reverse primer comprising primer sequences representedby SEQ ID NOs in Table 2 or homologs of SEQ ID NOs in Table 2 with atleast 80% more preferably 90%, still more preferably 91%, even morepreferably 92%, still more preferably 93%, even more preferably 94%,still more preferably 95%, even more preferably 96%, still morepreferably 97%, even more preferably 98%, still more preferably 99%, andmost preferably 99.8% or more identity and complementarity under similarstringency.

In one embodiment of the CocciENV assay, the forward primers compriseone or more degenerative primers. In another embodiment of the CocciENVassay, the reverse primers comprise one or more degenerative primers. Inyet another embodiment CocciENV assay, both the forward primers and thereverse primers comprise one or more degenerative primers. In someembodiments, the CocciENV assay may comprise more than 1 forward primerand more than 1 reverse primer. For example, the CocciENV assay maycomprise two, three, four and more primers; as such, the CocciENV assaymay comprise two forward primers and one reverse primer, or two forwardprimers and two reverse primers, or three forward primers and onereverse primer. In one embodiment, the CocciENV assay comprises aplurality of forward primers and a plurality of reverse primersrepresented by SEQ ID NOs: 10-38 (Table 2).

TABLE 2 CocciENV Assay Probe Name Probe Sequence 5′ to 3′ SEQ ID NOCQ_3_probe ACCCACATAGATTAGC SEQ ID NO: 2 Forward Primer Name ForwardPrimer Sequence 5′ to 3′ CocciEnv_F1d1 CGTTGCACRGGGAGCACCT SEQ ID NO: 10CocciEnv_F2 AAGCTTTGGATCTTTGTGGCTCT SEQ ID NO: 11 CocciEnv_F3AATTGATCCATTGCAAGCACCT SEQ ID NO: 12 CocciEnv_F4AATCCAACCTTTGGAACTACACCT SEQ ID NO: 13 CocciEnv_F5TTTTCCGGTATGGACTAGCACCT SEQ ID NO: 14 CocciEnv_F6d2TGTTAGGTAATCYAACYAGCACCT SEQ ID NO: 15 CocciEnv_F7d2TRTTAGGTAATYCAACTAGCACCT SEQ ID NO: 16 CocciEnv_F8d1TGTTAGATAATCCAACYAGCACCT SEQ ID NO: 17 CocciEnv_F9d2GKTARGTAATCCAACTAGCACCT SEQ ID NO: 18 CocciEnv_F10d2TGTTAGGTARTCCAACTAGCAYCT SEQ ID NO: 19 CocciEnv_F11d2TGTTAGGTAATCCAACTMGCACYT SEQ ID NO: 20 Reverse Primer Name ReversePrimer Sequence 5′ to 3′ CocciEnv_R1 GATGGAGGACTCTATATGCTTGT SEQ ID NO:21 CocciEnv_R2 ATGGAGGACTCGTTATGCCTGT SEQ ID NO: 22 CocciEnv_R3GGAGGACCCGTATGCTTGTGT SEQ ID NO: 23 CocciEnv_R4 TGCTAAATGATGGAGGGCTTGTSEQ ID NO: 24 CocciEnv_R5 GATGGAGGCTCGTATGCTTGT SEQ ID NO: 25CocciEnv_R6 AAGGGGTTTGTGGTGAATCCTTA SEQ ID NO: 26 CocciEnv_R7CAGAAAAATAGCCGTATGCTTGT SEQ ID NO: 27 CocciEnv_R8d2TRATGGAGRACTTGTATGCTTGT SEQ ID NO: 28 CocciEnv_R9d1TGATGGAGGACTCGTATGCYTGT SEQ ID NO: 29 CocciEnv_R10d2TGATGGARRACTCATATGCTTGT SEQ ID NO: 30 CocciEnv_R11d2TGATAGAGAACTTGTATRCTTRT SEQ ID NO: 31 CocciEnv_R12d2TGATGAAGAACTTRTATRCTTGT SEQ ID NO: 32 CocciEnv_R13d2TGATRRAGGACTTGTATGCTTGT SEQ ID NO: 33 CocciEnv_R14TGATGGAAAACTTGTATGCTTGT SEQ ID NO: 34 CocciEnv_R15d2TGATGGAGGACTTGTAYAYTTGT SEQ ID NO: 35 CocciEnv_R16d2TGATGGAGGACTTGTAYGCTTRT SEQ ID NO: 36 CocciEnv_R17d2TGATGGAGGACTYATATGCTTRT SEQ ID NO: 37 CocciEnv_R18d2GATGGAGGACTCGTWYGCTTGT SEQ ID NO: 38

Further illustrations of various aspects of the technology are detailedbelow.

II. Methods for Detecting Coccidioides Using Species Specific GenomicTarget Sequences

Methods that can be used to identify strain or species specific nucleicacids and alleles thereof, and biomarkers derived from transcriptionaland translational products of the strain or species specific nucleicacids and the alleles thereof, include PCR, Real-Time PCR,hybridization, sequencing and any combination of the above methods. Inone embodiment, the presence of the PCR or Real-Time PCR products in anassay may indicate the presence of Coccidioides species or one or morestrains thereof. In one embodiment, the PCR or Real Time-PCR productsmay be further identified or differentiated by hybridization performedeither simultaneously with or subsequently to the PCR reactions. Inanother embodiment, the PCR or Real-Time PCR products may be sequencedto ascertain the existence of a particular allele indicative of theidentity of Coccidioides species or one or more strains thereof in asample.

A nucleic acid may be added to a sample by any of a number of methods,including manual methods, mechanical methods, or any combinationthereof. The presence of the allele may be signified by any of a numberof methods, including amplification of a specific nucleic acid sequence,sequencing of a native or amplified nucleic acid, or the detection of alabel either bound to or released from the nucleic acid. Addition of thenucleic acid to the sample also encompasses a sample absent of thetarget allele to which the nucleic acid has specificity.

Nucleic acids may be selectively and specifically amplified from atemplate nucleic acid contained in a sample. In some nucleic acidamplification methods, the copies are generated exponentially. Examplesof nucleic acid amplification methods known in the art include:polymerase chain reaction (PCR), ligase chain reaction (LCR),self-sustained sequence replication (3 SR), nucleic acid sequence basedamplification (NASBA), strand displacement amplification (SDA),amplification with Qβ replicase, whole genome amplification with enzymessuch as φ29, whole genome PCR, in vitro transcription with Klenow or anyother RNA polymerase, or any other method by which copies of a desiredsequence are generated.

With PCR, it is possible to amplify a single copy of a specific targetsequence in genomic DNA to a level detectable by several differentmethodologies, such as hybridization with a labeled probe; incorporationof biotinylated primers followed by avidin-enzyme conjugate detection;incorporation of ³²P-labeled deoxynucleotide triphosphates—dCTP ordATP—into the amplified segment. In addition to genomic DNA, anyoligonucleotide or polynucleotide sequence can be amplified with anappropriate set of primer molecules. In particular, the amplifiedsegments created by the PCR process itself are, themselves, efficienttemplates for subsequent PCR amplifications.

PCR generally involves the mixing of a nucleic acid sample, two or moreprimers that are designed to recognize the template DNA, a DNApolymerase, which may be a thermostable DNA polymerase such as Taq orPfu, and deoxyribose nucleoside triphosphates (dNTPs). Reversetranscription PCR, quantitative reverse transcription PCR, andquantitative real time reverse transcription PCR are other specificexamples of PCR. In general, the reaction mixture is subjected totemperature cycles comprising a denaturation stage (typically 80-100°C.), an annealing stage with a temperature that is selected based on themelting temperature (Tm) of the primers and the degeneracy of theprimers, and an extension stage (for example 40-75° C.). In real-timePCR analysis, additional reagents, methods, optical detection systems,and devices known in the art are used that allow a measurement of themagnitude of fluorescence in proportion to concentration of amplifiedDNA. In such analyses, incorporation of fluorescent dye into theamplified strands may be detected or measured.

Alternatively, labeled probes that bind to a specific sequence duringthe annealing phase of the PCR may be used with primers. Labeled probesrelease their fluorescent tags during the extension phase so that thefluorescence level may be detected or measured. Generally, probes arecomplementary to a sequence within the target sequence downstream fromeither the upstream or downstream primer. Probes may include one or morelabel. A label may be any substance capable of aiding a machine,detector, sensor, device, or enhanced or unenhanced human eye fromdifferentiating a labeled composition from an unlabeled composition.Examples of labels include but are not limited to: a radioactive isotopeor chelate thereof, dye (fluorescent or nonfluorescent) stain, enzyme,or nonradioactive metal. Specific examples include, but are not limitedto: fluorescein, biotin, digoxigenin, alkaline phosphatese, biotin,streptavidin, ³H, ¹⁴C, ³²P, ³⁵S, or any other compound capable ofemitting radiation, rhodamine, 4-(4′-dimethylamino-phenylazo) benzoicacid (“Dabcyl”); 4-(4′-dimethylamino-phenylazo)sulfonic acid (sulfonylchloride) (“Dabsyl”); 5-((2-aminoethyl)-amino)-naphtalene-1-sulfonicacid (“EDANS”); Psoralene derivatives, haptens, cyanines, acridines,fluorescent rhodol derivatives, cholesterol derivatives;ethylenediaminetetraaceticacid (“EDTA”) and derivatives thereof or anyother compound that may be differentially detected. The label may alsoinclude one or more fluorescent dyes optimized for use in genotyping.Examples of dyes facilitating the reading of the target amplificationinclude, but are not limited to: CAL-Fluor Red 610, CAL-Fluor Orange560, dR110, 5-FAM, 6FAM, dR6G, JOE, HEX, VIC, TET, dTAMRA, TAMRA, NED,dROX, PET, BHQ+, Gold540, and LIZ.PCR.

Either primers or primers along with probes, as described above, willallow a quantification of the amount of specific template DNA present inthe initial sample. In addition, RNA may be detected by PCR analysis byfirst creating a DNA template from RNA through a reverse transcriptaseenzyme. In some aspects of the technology, the allele may be detected byquantitative PCR analysis facilitating genotyping analysis of thesamples.

An illustrative example, using dual-labeled oligonucleotide probes inPCR reactions is disclosed in U.S. Pat. No. 5,716,784 to DiCesare. Inthe PCR step of the multiplex Real Time-PCR/PCR reaction of the presenttechnology, the dual-labeled fluorescent oligonucleotide probe binds tothe target nucleic acid between the flanking oligonucleotide primersduring the annealing step of the PCR reaction. The 5′ end of theoligonucleotide probe contains the energy transfer donor fluorophore(reporter fluor) and the 3′ end contains the energy transfer acceptorfluorophore (quenching fluor). In the intact oligonucleotide probe, the3′ quenching fluor quenches the fluorescence of the 5′ reporter fluor.However, when the oligonucleotide probe is bound to the target nucleicacid, the 5′ to 3′ exonuclease activity of the DNA polymerase, e.g., TaqDNA polymerase, will effectively digest the bound labeledoligonucleotide probe during the amplification step. Digestion of theoligonucleotide probe separates the 5′ reporter fluor from the blockingeffect of the 3′ quenching fluor. The appearance of fluorescence by thereporter fluor is detected and monitored during the reaction, and theamount of detected fluorescence is proportional to the amount offluorescent product released. Examples of apparatus suitable fordetection include, e.g. Applied Biosystems™ 7900HT real-time PCRplatform (Applied Biosystems, Carlsbad, Calif.) and Roche's 480LightCycler (Roche, Basel, Switzerland), the ABI Prism 7700 sequencedetector (Applied Biosystems, Carlsbad, Calif.) using 96-well reactionplates or GENEAMP PC System 9600 or 9700 (Applied Biosystems, Carlsbad,Calif.) in 9600 emulation mode followed by analysis in the ABI PrismSequence Detector or TAQMAN LS-50B PCR Detection System (AppliedBiosystems, Carlsbad, Calif.). The labeled probe facilitated multiplexReal Time-PCR/PCR can also be performed in other real-time PCR systemswith multiplexing capabilities.

In some forms of PCR assays, quantification of a target in an unknownsample is often required. Such quantification is often in reference tothe quantity of a control sample. Generally, the control sample containsDNA at a known concentration. The control sample DNA may be a plasmidconstruct comprising only one copy of the amplification region to beused as quantification reference. To calculate the quantity of a targetin an unknown sample, various mathematical models are established.Calculations are based on the comparison of the distinct cycledetermined by various methods, e.g., crossing points (CP) and cyclethreshold values (Ct) at a constant level of fluorescence; or CPacquisition according to established mathematic algorithms.

The algorithm for Ct values in Real-Time PCR calculates the cycle atwhich individual PCR amplification reaches a significant threshold. Thecalculated Ct value is proportional to the number of target copiespresent in the sample, and the Ct value is a precise quantitativemeasurement of the copies of the target found in any sample. In otherwords, Ct values represent the presence of respective target that theprimer sets are designed to recognize. If the target is missing in asample, there should be no amplification in the Real Time-PCR reaction.

Alternatively, the Cp value may be utilized. A Cp value represents thecycle at which the increase of fluorescence is highest and where thelogarithmic phase of a PCR begins. The LightCycler® 480 Software (Roche,Basel, Switzerland) calculates the second derivatives of entireamplification curves and determines where this value is at its maximum.By using the second-derivative algorithm, data obtained are morereliable and reproducible, even if fluorescence is relatively low.

In addition to PCR, genotyping analysis may also be performed using aprobe that is capable of hybridizing to a nucleic acid sequence ofinterest. The term “hybridization” refers to the pairing ofcomplementary nucleic acids. Hybridization and the strength ofhybridization (i.e., the strength of the association between the nucleicacids) is impacted by such factors as the degree of complementaritybetween the nucleic acids, stringency of the conditions involved, the Tmof the formed hybrid, and the G:C ratio within the nucleic acids. Asingle molecule that contains pairing of complementary nucleic acidswithin its structure is said to be “self-hybridized.”

The terms “complementary” and “complementarity” refer to polynucleotides(i.e., a sequence of nucleotides) related by the base-pairing rules. Forexample, for the sequence “A-G-T,” is complementary to the sequence“T-C-A.” Complementarity may be “partial,” in which only some of thenucleic acids' bases are matched according to the base pairing rules.Or, there may be “complete” or “total” complementarity between thenucleic acids. The degree of complementarity between nucleic acidstrands has significant effects on the efficiency and strength ofhybridization between nucleic acid strands. This is of particularimportance in amplification reactions, as well as detection methods thatdepend upon binding between nucleic acids.

The term “homology” when used in relation to nucleic acids refers to adegree of complementarity. There may be partial homology, or completehomology and thus identical. “Sequence identity” refers to a measure ofrelatedness between two or more nucleic acids, and is given as apercentage with reference to the total comparison length. The identitycalculation takes into account those nucleotide residues that areidentical and in the same relative positions in their respective largersequences. Calculations of identity may be performed by algorithmscontained within computer programs such as “GAP” (Genetics ComputerGroup, Madison, Wis.) and “ALIGN” (DNAStar, Madison, Wis.). A partiallycomplementary sequence, one that at least partially inhibits (orcompetes with) a completely complementary sequence from hybridizing to atarget nucleic acid is referred to using the functional term“substantially homologous.” The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or Northern blot, solutionhybridization and the like) under conditions of low stringency. Asubstantially homologous sequence or probe will compete for and inhibitthe binding, or hybridization, of a sequence that is completelyhomologous to a target under conditions of low stringency. This is notto say that conditions of low stringency are such that non-specificbinding is permitted; low stringency conditions require that the bindingof two sequences to one another be a specific and selective interaction.The absence of non-specific binding may be tested by the use of a secondtarget which lacks even a partial degree of complementarity, forexample, less than about 30% identity); in the absence of non-specificbinding the probe will not hybridize to the second non-complementarytarget.

When used in reference to a double-stranded nucleic acid sequence suchas a cDNA or genomic clone, the term “substantially homologous” refersto any probe which can hybridize to either or both strands of thedouble-stranded nucleic acid sequence under conditions of low stringencyas described infra.

Low stringency conditions when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42° C. in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄.H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS,5×Denhardt's reagent (50×Denhardt's contains per 500 ml: 5 g Ficoll(Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)) and 100 μg/mldenatured salmon sperm DNA followed by washing in a solution comprising5×SSPE, 0.1% SDS at 42° C. when a probe of about 500 nucleotides inlength is employed.

High stringency conditions when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42° C. in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄.H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5×Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followedby washing in a solution comprising 0.1×SSPE, 1.0% SDS at 42° C. when aprobe of about 500 nucleotides in length is employed.

It is well known that numerous equivalent conditions may be employed tocomprise low stringency conditions; factors such as the length andnature (DNA, RNA, base composition) of the probe and nature of thetarget (DNA, RNA, base composition, present in solution or immobilized,etc.) and the concentration of the salts and other components, forexample, the presence or absence of formamide, dextran sulfate,polyethylene glycol, are considered and the hybridization solution maybe varied to generate conditions of low stringency hybridizationdifferent from, but equivalent to, the above listed conditions. Inaddition, conditions are known in the art that promote hybridizationunder conditions of high stringency, for example, increasing thetemperature of the hybridization and/or wash steps, the use of formamidein the hybridization solution, etc.

When used in reference to a double-stranded nucleic acid sequence suchas a cDNA or genomic clone, the term “substantially homologous” refersto any probe that can hybridize to either or both strands of thedouble-stranded nucleic acid sequence under conditions of low to highstringency as described above.

When used in reference to a single-stranded nucleic acid sequence, theterm “substantially homologous” refers to any probe that can hybridize,or is the complement of, the single-stranded nucleic acid sequence underconditions of low to high stringency as described above.

The term “Tm” refers to the “melting temperature” of a nucleic acid. Themelting temperature is the temperature at which a population ofdouble-stranded nucleic acid molecules becomes half dissociated intosingle strands. The equation for calculating the Tm of nucleic acids iswell known in the art. As indicated by standard references, a simpleestimate of the Tm value may be calculated by the equation:Tm=81.5+0.41(% G+C), when a nucleic acid is in aqueous solution at 1 MNaCl (See for example, Anderson and Young, Quantitative FilterHybridization (1985) in Nucleic Acid Hybridization). Other referencesinclude more sophisticated computations that take structural as well assequence characteristics into account for the calculation of Tm.

As used herein the term “stringency” refers to the conditions oftemperature, ionic strength, and the presence of other compounds such asorganic solvents, under which nucleic acid hybridizations are conducted.With “high stringency” conditions, nucleic acid base pairing will occuronly between nucleic acid fragments that have a high frequency ofcomplementary base sequences. Thus, conditions of “low” stringency areoften required with nucleic acids that are derived from organisms thatare genetically diverse, as the frequency of complementary sequences isusually less.

Probes for hybridization may comprise nucleic acids, oligonucleotides(DNA or RNA), proteins, protein complexes, conjugates, natural ligands,small molecules, nanoparticles, or any combination of molecules thatincludes one or more of the above, or any other molecular entity capableof specific binding to any allele, whether such molecular entity existsnow or is yet to be disclosed. In one aspect of the technology, theprobe comprises an oligonucleotide, as described herein.

Under some circumstances, methods of detecting a gene or an allele mayinvolve assessing their expression level through their transcriptionalor translational products such as a RNA or protein molecules. Theexpression of a gene or an allele may be assessed by any of a number ofmethods used currently in the art and yet to be developed. Examplesinclude any nucleic acid detection method, including the followingnon-limiting examples, microarray analysis, RNA in situ hybridization,RNAse protection assay, Northern blot. Other examples include anyprocess of detecting expression that uses an antibody including thefollowing non-limiting examples, flow cytometry, immunohistochemistry,ELISA, Western blot, Northwestern blot, and immunoaffinitychromatograpy. Antibodies may be monoclonal, polyclonal, or any antibodyfragment, for example, Fab, F(ab)₂, Fv, scFv, phage display antibody,peptibody, multi-specific ligand, or any other reagent with specificbinding to a target. Other methods of assessing protein expressioninclude the following non-limiting examples: HPLC, mass spectrometry,protein microarray analysis, PAGE analysis, isoelectric focusing, 2-Dgel electrophoresis, and enzymatic assays.

In some aspects of the technology, the presence of an allele may beestablished by binding to probes in a media or on a microarray such as aDNA chip. Examples of DNA chips include chips in which a number ofsingle stranded oligonucleotide probes are affixed to a solid substratesuch as silicon glass. Oligonucleotides with a sequence complementary toan allele are capable of specifically binding to that allele to theexclusion of alleles that differ from the specific allele by one or morenucleotides. Labeled sample DNA is hybridized to the oligonucleotidesand detection of the label is correlated with binding of the sample, andconsequently, the presence of the allele in the sample.

In allele-specific hybridization, oligonucleotide sequences representingall possible variations at a polymorphic site are included on a chip.The chip and sample are subjected to conditions under which the labeledsample DNA will bind only to an oligonucleotide with an exact sequencematch. In allele-specific primer extension, sample DNA hybridized to thechip may be used as a synthesis template with the affixedoligonucleotide as a primer. Under this method, only the added dNTPs arelabeled. Incorporation of the labeled dNTP then serves as the signalindicating the presence of the allele. The fluorescent label may bedetected by any of a number of instruments configured to read at leastfour different fluorescent labels on a DNA chip. In another alternative,the identity of the final dNTP added to the oligonucleotide may beassessed by mass spectrometry. In this alternative, the dNTP's may, butneed not be labeled with a label of known molecular weight.

A nucleic acid probe may be affixed to a substrate. Alternatively, asample may be affixed to the substrate. A probe or sample may becovalently bound to the substrate or it may be bound by somenon-covalent interaction including electrostatic, hydrophobic, hydrogenbonding, Van Der Waals, magnetic, or any other interaction by which aprobe such as an oligonucleotide probe may be attached to a substratewhile maintaining its ability to recognize the allele to which it hasspecificity. A substrate may be any solid or semi-solid material ontowhich a probe may be affixed, either singly or in the presence of one ormore additional probes or samples as is exemplified in a microarray.Examples of substrate materials include but are not limited topolyvinyl, polystyrene, polypropylene, polyester or any other plastic,glass, silicon dioxide or other silanes, hydrogels, gold, platinum,microbeads, micelles and other lipid formations, nitrocellulose, ornylon membranes. The substrate may take any form, including a sphericalbead or flat surface. For example, the probe may be bound to a substratein the case of an array or an in situ PCR reaction. The sample may bebound to a substrate in the case of a Southern Blot.

A nucleic acid probe may include a label. A label may be any substancecapable of aiding a machine, detector, sensor, device, or enhanced orunenhanced human eye from differentiating a labeled composition from anunlabeled composition. Examples of labels include, but are not limitedto: a radioactive isotope or chelate thereof, dye (fluorescent ornon-fluorescent) stain, enzyme, or nonradioactive metal. Specificexamples include, but are not limited to: fluorescein, biotin,digoxigenin, alkaline phosphatase, biotin, streptavidin, ³H, ¹⁴C, ³²P,³⁵S, or any other compound capable of emitting radiation, rhodamine,4-(4′-dimethylamino-phenylazo)benzoic acid (“Dabcyl”);4-(4′-dimethylamino-phenylazo)sulfonic acid (sulfonyl chloride)(“Dabsyl”); 5-((2-aminoethyl)-amino)-naphtalene-1-sulfonic acid(“EDANS”); Psoralene derivatives, haptens, cyanines, acridines,fluorescent rhodol derivatives, cholesterol derivatives;ethylenediaminetetraaceticacid (“EDTA”) and derivatives thereof, or anyother compound that may be differentially detected. The label may alsoinclude one or more fluorescent dyes optimized for use in genotyping.Examples of such dyes include, but are not limited to: dR110, 5-FAM,6FAM, dR6G, JOE, HEX, VIC, TET, dTAMRA, TAMRA, NED, dROX, PET, BHQ+,Gold540, and LIZ. In one embodiment, the probe comprising SEQ ID NO: 2is labeled with 6FAM at 5′ end and MGB-NFQ at 3′ end.

Methods of detecting the presence of a gene or an allele furtherinclude, but are not limited to, any form of DNA sequencing includingSanger, next generation sequencing, pyrosequencing, sequencing byligation, sequencing by synthesis, single molecule sequencing, pooled,and barcoded DNA sequencing or any other sequencing method now known oryet to be disclosed; or any other method that allows the detection of aparticular nucleic acid sequence within a sample or enables thedifferentiation of one nucleic acid from another nucleic acid thatdiffers from the first nucleic acid by one or more nucleotides, or anycombination of these.

In Sanger Sequencing, a single-stranded DNA template, a primer, a DNApolymerase, nucleotides and a label such as a radioactive labelconjugated with the nucleotide base or a fluorescent label conjugated tothe primer, and one chain terminator base comprising a dideoxynucleotide(ddATP, ddGTP, ddCTP, or ddTTP) are added to each of four reactions (onereaction for each of the chain terminator bases). The sequence may bedetermined by electrophoresis of the resulting strands. In dyeterminator sequencing, each of the chain termination bases is labeledwith a fluorescent label of a different wavelength which allows thesequencing to be performed in a single reaction.

In pyrosequencing, the addition of a base to a single stranded templateto be sequenced by a polymerase results in the release of aphyrophosphate upon nucleotide incorporation. An ATP sulfurylase enzymeconverts pyrophosphate into ATP which, in turn, catalyzes the conversionof luciferin to oxyluciferin which results in the generation of visiblelight that is then detected by a camera.

In sequencing by ligation, such as, SOLID™ sequencing, the molecule tobe sequenced is fragmented and used to prepare a population of clonalmagnetic beads, in which each bead is conjugated to a plurality ofcopies of a single fragment with an adaptor sequence, and alternatively,a barcode sequence. The beads are bound to a glass surface. Sequencingis then performed through 2-base encoding.

In sequencing by synthesis, randomly fragmented targeted DNA is attachedto a surface. The fragments are extended and bridge amplified to createa flow cell with clusters, each with a plurality of copies of a singlefragment sequence. The templates are sequenced by synthesizing thefragments in parallel. Bases are indicated by the release of afluorescent dye correlating to the addition of the particular base tothe fragment.

III Kits.

Kits that facilitate methods of detecting a strain or species specificsequence may include one or more of the following reagents: specificnucleic acids such as oligonucleotides, labeling reagents, enzymesincluding PCR amplification reagents such as the thermostable DNApolymerases Taq or Pfu, reverse transcriptase, or one or more otherpolymerases, and/or reagents that facilitate hybridization. Specificnucleic acids may include nucleic acids, polynucleotides,oligonucleotides (DNA, or RNA), or any combination of molecules thatincludes one or more of the above, or any other molecular entity capableof specific binding to a nucleic acid marker. In one aspect of thetechnology, the specific nucleic acid comprises one or moreoligonucleotides capable of hybridizing to the marker.

A kit may also contain an indication that links the output of the kit toa particular result. For example, an indication may be one or moresequences or that signify the identification of a particular fungalphylum, class, order, family, genus species, subspecies, strain or anyother delineation of a group of fungi. An indication may include a Ctvalue, wherein exceeding the Ct value indicates the presence or absenceof an organism of interest. A kit may contain a positive control. A kitmay contain a standard curve configured to quantify the amount of funguspresent in a sample. An indication includes any guide that links theoutput of the kit to a particular result. The indication may be a levelof fluorescence or radioactive decay, a value derived from a standardcurve, or from a control, or any combination of these and other outputs.The indication may be printed on a writing that may be included in thekit or it may be posted on the Internet or embedded in a softwarepackage.

EXAMPLES

Various embodiments of the present teachings can be illustrated by thefollowing non-limiting examples. The following embodiments and examplesare illustrative, and are not intended to limit the scope of the claims.

Example 1 Method and Material—CocciDxQ

The assay employs TaqMan MGB-6FAM fluorescent probe and a multiplex setof three forward primers and 4 reverse primers (Table 1 above). Theassay reactions can be performed using Real Time PCR Mastermix ofchoice, but has been optimized for use with Quanta Biosciences PerfeCTa®qPCR FastMix®, UNG, ROX™. Thermocycling conditions consist of UNGactivation for 3 min at 50° C. followed by 10 min Taq Polymeraseactivation at 95° C. and 50 PCR cycles of 15 s at 95° C. and 1 min at60° C. Each reaction produced an amplification plot yielding acycle-threshold (Ct) value directly proportional to the initialconcentration of DNA in the reaction.

Example 2 Sensitivity and Specificity of the Cocci QuantitativeDiagnosis Assay—CocciDxQ

(1) Determining Limit of Detection

The Limit of Detection (LOD), also called the Detection Limit or LowerLimit of Detection, is the lowest quantity of a substance that can bedistinguished from the absence of that substance (i.e., a blank value)within a stated confidence limit. LOD is hereby used to describe thesensitivity of quantitative assays. The assay target region, amulti-copy target having the advantage of being detected at low levelsin comparison to a single-copy target was utilized in the LOD test.Although the copy number of assay target region in Coccidioidesisolates, including C. immitis and C. posadasii, varies, however, theaverage number of target copies in a Coccidioides genome is estimated at85. Therefore, the ability of an assay in detecting the target regionprovides a method for relative quantification of Coccidioides fungalload.

The analytical LOD of the CocciDxQ assay is 15 target copies/ul (Table3). This translates to less than one genome/ul. Genomic DNA wasquantified and limiting serial dilutions were created to test the LOD.Dilutions were queried across the CocciDxQ assay with 20 replicateseach. Finally, to establish the LOD, dilutions for which at least 19 of20 replicates amplified were further evaluated by testing 64 replicatesand exhibited at least 95% amplification (61/64 amplification ratio).Results are shown in Table 3.

TABLE 3 Determination of Limit of Detection of CocciDxQ assay: CocciDxQLimit Dilution Amplification Amplification of Detection of Target Ratioof 20 Ratio of 64 (Target Copies/ Replicate Replicate Mean Copies/ 1 ulScreen Screen Ct 1 ul) 25 20/20 62/64 31.08 15 copies/ 15 20/20 62/6431.72 1 μl 10 19/20 58/64 32.61 (Ct = 5 17/20 N/A 36.93 31.72, 3 14/20N/A 37.74 Ct std. 1  6/20 N/A 37.80 dev. = 0.1  2/20 N/A 36.45 0.77)0.01  3/20 N/A 38.08

The analytical limit of detection of the assay is 15 target copies/ul,that means if the copy number/1 μl of the genomic target in a sample islower than 15, the CocciDxQ assay may not be sensitive enough to eitherreliably detect the presence or absence of the target, nor a reliablecalculation of the copy number of a target DNA in the sample. However,the sensitivity of the CocciDxQ assay is imparted by high genomic copynumber of the target region, an area associated with a copia-likeretrotransposon, which is 85 copies/genome. That is to say that theCocciDxQ assay as disclosed herein can detect equivalent to less thanone genomic DNA molecule per microliter of DNA, which is highlysensitive.

(2) Assay Specificity

To further illustrate the specificity of the CocciDxQ assay, the assaywas tested against a panel of 89 diagnostic differential DNA's includingdifferential diagnostic isolates and near neighbor or backgroundisolates to detect any cross reactivity. All assay results were negative(see Table 4), indicating the sample species does not contain C. immitisand C. posadasii specific sequence amplifiable using the CocciDxQ assaycomprising probe and primer sets in Table 1, and thus proved the assayspecificity.

TABLE 4 List of DNA that the CocciDxQ Assay was screened across. HumangDNA Streptococcus pneumoniae Burkholderia pseudomallei Staphylococcuscapitis Streptococcus lactis Mycoplasma pneumoniae Streptococcus oralisEnterobacter cloacae Haemophilus Influenzae Streptococcus mitisAcinetobacter baumanni Streptococcus salivarius Streptococcusthermophilus Methicillin Resistant Staphylococcus aureus Streptococcusanginosus Methicillin Sensitive Staphylococcus aureus Streptococcusmutans Micrococcus sp Staphylococcus arlettae Chryseobacteriumindologenes Staphylococcus chonii Klebsiella oxytoca Staphylococcusequorum Enterococcus faecalis Staphylococcus gallinarum Haemophilusparainfluenzae Staphylococcus hominis Achromobacter xylosoxidansStaphylococcus kloosii Staphylococcus xylosus Staphylococcus lugdunensisKlebsiella pneumoniae Streptococcus gordonii Moraxella catarrhalisStreptococcus equi Staphylococcus epidermidis Streptococcus uberisStaphylococcus haemolyticus Providencia stuartii Streptococcus pyogenesCorynebacterium jeikeium Acremonium strictum Stenotrophomonasmaltophilia Bacillus anthracis Fusobacterium nucleatum Brucella abortusCorynebacterium diphtheriae Candida famata Porphyromonas gingivalisCandida haemulonii Cryptococcus neoformans Candida lusitaniaeMycobacterium avium Chaetomium globosum Aspergillus niger Escherichacoli Penicillium marneffei Francisella tularensis Eikenella corrodensFusarium solani Enterobacter aerogenes Geotrichum candidumStaphylococcus saprophyticus Histoplasma capsulatum Pseudomonasaeruginosa Legionella pneumophila Neisseria meningitidis Listeriamonocytogenes Entercoccus faecium Paecilomyces variotii Neisseriagonorrhoeae Pichia ohmeri Burkholderia cepacia Rhizopus oryzaeBordetella bronchiseptica Salmonella typhimurium Candida albicansSporothrix schenckii Bacteroides fragilis Trichosporon asteroidesBacteroides uniformis Trichosporon faecale Streptococcus agalactiaeTrichosporon ovoides Candida glabrata Uncinocarpus reesi Candidaparapsilosis Burkholderia ubonensis Candida tropicalis

The CocciDxQ assay was further screened across samples containingCoccidioides spp. using DNA extracts or whole genome amplifications ofDNA extracts, and the assay detected Coccidioides spp. in 559 out of atotal number of 560 samples.

Example 3 CocciDxQ Assay for Clinical Specimen

Clinical specimen suspected having Coccidioides spp. were tested withthe CocciDxQ assay. DNA was extracted from specimens which were blood,sputum, saliva, urine, or sputum-LSA. The test results provided in Table5 show that sputum samples provide template (e.g., DNA) suitable for theCocciDxQ assay.

TABLE 5 CocciDxQ test using DNA of clinical samples Amplification rateSpecimen Type (# of samples tested) Mean Ct Blood 0 (13) n/a Sputum 1(6) 37.2 Saliva 0 (14) n/a Urine 0 (13) n/a Sputum-LSA 16 (25) 27.5

DNA and RNA extracted from pleural fluid specimens were also testedusing the CocciDxQ assay. The Real-Time PCR results are shown in Table6.

TABLE 6 CocciDxQ assay for clinical pleural fluid specimens SampleCocciDxQ Ct on DNA CocciDxQ Ct on RNA 3838H Neg 38.0 0681J Neg 37.18056G Neg Neg 7477G Neg Neg 9294H 35.1 37.6 9496G Neg Neg 5308G Neg NegNeg = negative for target

The results from Table 6 illustrate that RNA can also be used as anassay target/template, in addition to DNA, if a reverse transcriptionstep is used to generate cDNA. Further, Coccidioides was detected inseveral samples that had negative detection results in DNA. Thus, theseresults could demonstrate that RNA detection of Coccidioides can be usedin addition to, or in place of DNA-based detection of Coccidioides.

Another set of clinical specimen were tested with the CocciDxQ assayusing both DNA and RNA from each specimen, and the results are providedin Table 7:

TABLE 7 CocciDxQ assay for clinical specimen DNA RNA ComparisonComparison Comparison real-time Control real-time real-time assays toreal-time assays to assays to ITS PCR ITS ITS Sample Name CDxQ CQ34 CQBD16S ALU CDxQ CQ34 CQBD 16S ALU TG004-2_saliva Neg Neg Neg 30.7 NR NegNeg Neg 21.0 25.4 TG006_saliva Neg Neg Neg 31.5 19.6 Neg Neg Neg 26.222.3 TG006-2_saliva Neg Neg Neg 28.7 20.7 Neg Neg Neg 25.9 23.7TG009_saliva Neg Neg Neg 29.9 17.6 Neg Neg Neg 22.2 20.0 TG009-2_salivaNeg Neg Neg 26.1 20.5 Neg 38.6 Neg 24.4  7.3 TG010_saliva Neg Neg Neg31.3 21.0 Neg Neg Neg 23.7 20.1 TG010-2_saliva Neg Neg Neg 32.1 21.0 NegNeg Neg 27.1 18.8 TG010-2_sputum Neg Neg Neg 32.1 NR Neg Neg Neg 31.616.1 TG011_sputum Neg Neg Neg 24.4 NR Neg Neg Neg 25.0 18.4 TG012_salivaNeg Neg Neg 31.7 21.6 Neg Neg Neg 29.2 24.7 TG012-2_saliva Neg Neg Neg31.8 17.3 Neg Neg Neg 28.7 19.6 TG012-2_sputum Neg Neg Neg 28.7 NR NegNeg Neg Neg 31.7 TG012-3_saliva Neg Neg Neg 31.8 20.3 Neg Neg Neg 27.621.2 TG013_sputum 38.2 37.7 Neg 30.8 26.4 27.8 25.3 Neg 28.3 19.0TG013_saliva Neg Neg Neg 24.4 NR Neg Neg Neg 24.8 19.0 TG013_sputum NegNeg Neg 24.4 NR 36.9 35.1 Neg 16.2 12.9 TG014_saliva Neg Neg Neg 31.9 NRNeg Neg Neg 27.9 34.4 TG015_saliva Neg Neg Neg 30.8 21.0 Neg Neg Neg25.3 21.6 TG015-2_sputum Neg Neg Neg 23.7 NR Neg Neg Neg 21.5 24.8TG016_sputum Neg Neg Neg 21.6 NR Neg Neg Neg 16.4 12.8 PC (DNA only)17.3 20.9 20.4 9.9 NR NR NR NR NR NR

Example 4 CocciENV

With the discovery of new alleles of SEQ ID NO: 1 (i.e., the targetsequence of CocciDxQ and CocciENV), additional oligonucleotides (SEQ IDNOs: 10-38) were generated to bind to and amplify some or all of theknown alleles of SEQ ID NO: 1. In brief, Ccpy numbers of the targetsequence (SEQ ID NO: 1) in Coccidioides genomes were estimatedbioinformatically using whole genome sequence with this updatedinformation. Using the Taqman probe sequence (SEQ ID NO: 2), each genomewas queried via BLAST for hits with 100% identity and 100% coverage. Foreach hit, the probe and flanking sequence were extracted and aligned toconfirm the region's homology to the assay target (SEQ ID NO: 1).

The CocciENV assay is configured to be run as a real-time PCR reactionusing substantially similar conditions as recited above for the CocciDxQassay, but with modifications as to the oligonucleotide content. Inshort, the CocciENV assay uses the same probe (SEQ ID NO: 2), butreplaces the CocciDxQ forward and reverse primers (SEQ ID NOs: 3-9) withthe forward and reverse primers detailed in Table 2 (SEQ ID NOs: 10-38).

The CocciEnv assay was subject to concise validation, given theextensive validation of CocciDxQ (described above), but includedsensitivity and specificity screening across a subset of the targetmolecules mentioned above, and added DNA from four additional Onygenalesspecies that are more phylogenetically closely related to Coccidioidesspp: Amauroascus mutatus, A. niger, Byssoonygena ceratinophila, andChrysosporium queenslandicum.

Example 5 CocciENV Development and Validation A. Methods

With the recent database deposition of new Coccidioides genomesequences, we hypothesized that more variant alleles of the CocciDxQtarget would be available, and that we could add primers to the originalassay to capture more variants of the target, thus increasing thesensitivity of the assay. Using a local BLAST database of all availableCoccidioides genomes and the CocciDxQ Taqman probe sequence as a query,each genome was queried for hits with 100% identity and 100% coverage.For each hit, the probe and flanking sequence were extracted using anin-house script and aligned to confirm the region's identity to theassay target. We designed several new primers to increase the number ofalleles of the target captured by the assay, and refer to the enhancedassay as CocciEnv. The new assay was run using the same conditions asfor CocciDxQ, with only primer concentrations modified (Table 8).

TABLE 8 Assay SEQ ID Final concentration component Sequence NO: in PCR(uM) CDx_F1d1 CGTTGCACRGGGAGCACCT 10 0.375 CDx_F2AAGCTTTGGATCTTTGTGGCTCT 11 0.375 CDx_F3 AATTGATCCATTGCAAGCACCT 12 0.25CDx_F4 AATCCAACCTTTGGAACTACACCT 13 0.25 CDx_F5 TTTTCCGGTATGGACTAGCACCT14 0.375 CDx_F6d2 TGTTAGGTAATCYAACYAGCACCT 15 0.125 CDx_F7d2TRTTAGGTAATYCAACTAGCACCT 16 0.125 CDx_F8d1 TGTTAGATAATCCAACYAGCACCT 170.125 CDx_F9d2 GKTARGTAATCCAACTAGCACCT 18 0.125 CDx_F10d2TGTTAGGTARTCCAACTAGCAYCT 19 0.125 CDx_F11d2 TGTTAGGTAATCCAACTMGCACYT 200.125 CDx_R1 GATGGAGGACTCTATATGCTTGT 21 0.375 CDx_R2ATGGAGGACTCGTTATGCCTGT 22 0.375 CDx_R3 GGAGGACCCGTATGCTTGTGT 23 0.375CDx_R4 TGCTAAATGATGGAGGGCTTGT 24 0.375 CDx_R5 GATGGAGGCTCGTATGCTTGT 250.375 CDx_R6 AAGGGGTTTGTGGTGAATCCTTA 26 0.375 CDx_R7CAGAAAAATAGCCGTATGCTTGT 27 0.375 CDx_R8d2 TRATGGAGRACTTGTATGCTTGT 280.125 CDx_R9d1 TGATGGAGGACTCGTATGCYTGT 29 0.125 CDx_R10d2TGATGGARRACTCATATGCTTGT 30 0.125 CDx_R11d2 TGATAGAGAACTTGTATRCTTRT 310.125 CDx_R12d2 TGATGAAGAACTTRTATRCTTGT 32 0.125 CDx_R13d2TGATRRAGGACTTGTATGCTTGT 33 0.125 CDx_R14 TGATGGAAAACTTGTATGCTTGT 340.125 CDx_R15d2 TGATGGAGGACTTGTAYAYTTGT 35 0.125 CDx_R16d2TGATGGAGGACTTGTAYGCTTRT 36 0.125 CDx_R17d2 TGATGGAGGACTYATATGCTTRT 370.125 CDx_R18d2 GATGGAGGACTCGTWYGCTTGT 38 0.125 CDx_FMGB6FAM-ACCCACATAGATTAGC-MGBNFQ 2 0.25

CocciEnv was subject to a more concise validation than described above,given the extensive validation of CocciDxQ, but included sensitivity andspecificity screening across a subset of the DNAs used for CocciDxQvalidation (n=94 Coccidioides WGA DNAs, n=89 non-target DNAs Table 9),along with DNA from four additional Onygenales species: Amauroascusmutatus ATCC® 90275, A. niger ATCC® 22339, Byssoonygena ceratinophilaATCC® 64724, and Chrysosporium queenslandicum ATCC® 4404. Additionally,CocciDxQ and CocciEnv were compared side-by-side by screening DNAs from23 Coccidioides isolates.

B. Results

The CocciDxQ assay was positive on the whole-genome-amplified DNA of all556 unique isolates of Coccidioides (Table 10), and was negative on allDNA from various species (Table 9) including the four Onygenales familymembers, illustrating 100% sensitivity and 100% specificity. Theabbreviated validation showed that CocciEnv also demonstrated 100%sensitivity and specificity.

TABLE 9 Isolate gDNAs screened to confirm specificity of CocciDxQ andCocciEnv assays. Species ID Strain ID Human Streptococcus lactis 22cStreptococcus oralis 22d Haemophilus Influenzae PU5-052 Acinetobacterbaumanni ACBA-3 Streptococcus thermophilus BAA-250 Streptococcusanginosus 33397 Streptococcus mutans 700610 Staphylococcus arlettae43957 Staphylococcus chonii 29974 Staphylococcus equorum 43958Staphylococcus gallinarum 35539 Staphylococcus hominis 27844Staphylococcus kloosii 43959 Staphylococcus lugdunensis 43809Streptococcus gordonii 10558 Streptococcus equi 9528 Streptococcusuberis 700407 Providencia stuartii PROV-1 Corynebacterium jeikeium jkgrp COJE-1 Stenotrophomonas maltophilia STMA-10 Fusobacterium nucleatumATCC25586D-5 Corynebacterium diphtheriae  ATCC700971D-5 Porphyromonasgingivalis ATCC33277D-5 Cryptococcus neoformans  ATCC208821D-2Mycobacterium avium BAA-968D-5 Aspergillus niger 1015D-2 Penicilliummarneffei 18224-D2 Eikenella corrodens 51724D Enterobacter aerogenes15038D-5 Pseudomonas aeruginosa PSAR-64 Neisseria meningitidis CRS8-001Entercoccus faecium VRE-33 Neisseria gonorrhoeae CRS6-374 Burkholderiacepacia ATCC 25608 Bordetella bronchiseptica ATCC 10580 Candida albicansATCC 14053 Bacteroides fragilis ATCC 25285 Bacteroides uniformis ATCC8492  Streptococcus agalactiae CRS4-147 Candida glabrata YT-48 Candidaparapsilosis YT-49 Candida tropicalis YT-50 Streptococcus pneumoniaeSTPN-187 Staphylococcus capitis CNS-125 Mycoplasma pneumoniae 15531Enterobacter cloacae CRS4-429 Streptococcus mitis STMI-1 Streptococcussalivarius SSAL-1 Methicillin Resistant Staphylococcus aureus MRSA-653Methicillin Sensitive Staphylococcus aureus MSSA-309 Micrococcus spMIC-3 Chryseobacterium indologenes CHIN-8 Klebsiella oxytoca KOXY-142Enterococcus faecalis EFA-115 Haemophilus parainfluenzae HPAR-304Achromobacter xylosoxidans ACXY-2 Staphylococcus xylosus ATCC-35033Klebsiella pneumoniae SP-1237, KLPN-143 Moraxella catarrhalis MCAT-108Staphylococcus epidermidis HIP04645 Staphylococcus haemolyticus N/AStreptococcus pyogenes GAS-143 Coccidioides posadasii 3224 Coccidioidesposadasii 3231 Acremonium strictum Candida famata Candida haemuloniiCandida lusitaniae Chaetomium globosum Coccidioides immitis Escherichacoli 0157:H7 ATCC35150 Francisella tularensis LVS Fusarium solaniGeotrichum candidum Histoplasma capsulatum Legionella pneumophila ATCC33152 Listeria monocytogenes H2446 Paecilomyces variotii Pichia ohmeriRhizopus oryzae Salmonella typhimurium LT1 Sporothrix schenckiiTrichosporon asteroides Trichosporon faecale Trichosporon ovoidesUncinocarpus reesi Yersinia pestis FV-1 Burkholderia ubonensis NCTC13147Amauroascus mutatus Amauroascus niger Byssoonygena ceratinophilaChrysosporium queenslandicum

TABLE 10 Isolate gDNA (whole genome amplified) screened with CocciDxQassay. A subset of these samples (n = 94) was screened with CocciEnvassay. For origin, C = clinical and E = environmental. Isolation/DiseaseTGC ID# Species ID Strain ID RMSCC#/Alternate ID Information SourceOrigin TGC0001 C. posadasii 8178 ID05- 2440008178, KJK004 Bronchial washC TGC0002 C. posadasii 8533 ID05- 2560008533, KJC010 Bronchial wash CTGC0003 C. posadasii 8589 ID05- 2570008589, KJB010 Tissue, left cheek CTGC0004 C. posadasii 63029 bronch wash TGC0005 C. posadasii 8700 ID05-2620008700, KJA011 Ankle tissue C TGC0006 C. posadasii 8835 ID05-2650008835, KJA012e Bronchial wash C TGC0008 C. posadasii 63394 R. lungwash TGC0009 C. posadasii 8885 ID05- 2690008885, KJK008f Blood C TGC0010C. posadasii 8860 Unknown TGC0011 C. posadasii 8973 ID05- 2700008973,KJA014g Bone marrow C TGC0012 C. posadasii 9001 ID05- 2710009001, KJB011Sputum C TGC0013 C. posadasii 9120 ID05-2760009120 Blood C TGC0015 C.posadasii 63363 bronch wash TGC0016 C. posadasii 63360 bronch washTGC0017 C. posadasii 63078 wash/aspirate TGC0018 C. posadasii 10512bronch wash TGC0019 C. posadasii 10569 sputum TGC0020 C. posadasii 10816CSF TGC0021 C. posadasii 10995 bronch wash TGC0022 C. posadasii 10997sputum TGC0023 C. posadasii 11166 LUL BAL TGC0024 C. posadasii 11206tissue TGC0025 C. posadasii 969 ID05- 0340000969, KJE001 Bronchial washC TGC0026 C. posadasii 152 aspirate TGC0027 C. posadasii 150 sputumTGC0028 Unknown 151 swab TGC0029 C. posadasii 3718 RML wash TGC0030 C.posadasii 3601 BAL RLL TGC0031 C. posadasii 94 body fluid TGC0032 C.immitis 3698 RMSCC 3698 ~ ~ TGC0033 C. posadasii 15 sputum TGC0034 C.posadasii 11224 sputum TGC0035 C. posadasii 11226 sputum TGC0036 C.posadasii 11223 bronch wash TGC0037 C. posadasii 3632 right lung noduleTGC0038 C. posadasii 416 ID05- 0180000416, KJD001 Bronchial wash CTGC0039 C. posadasii 678 ID05- 0250000678, KJC002 Bronchial wash CTGC0040 C. posadasii 295 ID05- 0120000295, KJC001 Elbow fluid C TGC0041C. posadasii 3621 sputum TGC0042 C. posadasii 276 ID05-0110000276 Kneefluid C TGC0043 C. posadasii 153 tissue TGC0044 C. posadasii 2928 ID05-0750002928, KJA003 Bronchial wash C TGC0045 C. posadasii 3402 ID05-08200003402, KJF002 Elbow tissue C TGC0046 Unknown 1628 sputum TGC0047C. posadasii 2870 ID05- 0740002870, KJA002 Bronchial wash C TGC0048 C.posadasii 1246 ID05- 0420001246, KJC003 Bronchial wash C TGC0049 C.posadasii 10996 bronch wash TGC0050 C. posadasii 62461 Leukens Trap (?)TGC0051 C. posadasii 93 Unknown TGC0052 C. posadasii 61107 wash/aspirateTGC0053 C. posadasii 61833 Unknown TGC0054 C. posadasii 5521 BAL RLLTGC0055 C. posadasii 06-920  RLLL tissue TGC0056 C. posadasii 11492 neckmass TGC0057 C. posadasii 5804 ID05- 1650005804, KJA008 Bronchial wash CTGC0058 C. posadasii 62248 RUL wash TGC0059 Unknown 3761 bronch washTGC0060 C. posadasii 06-1110 unknown TGC0061 C. posadasii 4171 ID05-1100004171, KJB004 Bronchial wash C TGC0062 C. posadasii 61961 sacralmass TGC0063 C. posadasii 62165 lung tissue TGC0064 C. posadasii 4916ID05- 13300004916, Bronchial wash C KJE002b TGC0065 Unknown 2219 CSFTGC0066 C. posadasii 2454 L arm node TGC0067 C. posadasii 3399paratracheal node TGC0068 C. posadasii 3359 f0702 Pleural fluid CTGC0069 Unknown 61312 CSF TGC0070 C. posadasii 62962 bronch RML TGC0071C. posadasii 62987 bronch wash TGC0072 C. posadasii 62868 bronch washTGC0073 C. posadasii 61948 bronch wash TGC0074 C. posadasii 62882 lungfluid TGC0075 C. posadasii 11414 bronch wash TGC0076 C. posadasii 61109unknown swab TGC0077 C. posadasii 61855 lung-right TGC0078 C. posadasii61812 BAL TGC0079 C. posadasii 61859 bronch wash TGC0080 C. posadasii61414 unknown TGC0081 C. posadasii 5523 ID05- 1540005523, KJB007cBronchial wash C TGC0082 C. posadasii 5579 ID05- 1570005579, KJA007Bronchial wash C TGC0083 C. posadasii 6961 bronch wash TGC0084 C.posadasii 6840 BAL TGC0085 C. posadasii 62215 bronch wash TGC0086 C.posadasii 62628 bronch wash TGC0087 C. posadasii 1024 ID05- 0380001024,KJF001 Knee Fluid C TGC0088 C. posadasii 3453 Trans trachea C TGC0089 C.posadasii 1130 ID05- 0400001130, KJD002 Knee Fluid C TGC0090 C.posadasii 1298 ID05- 04200001298, KJB003 Bronchial wash C TGC0091 C.posadasii 3833 ID05-0970003833, KJA004 Bronchial wash C TGC0092 C.posadasii 3614 ID05- 0890003614, KJC004 Lung Tissue C TGC0093 C.posadasii 4000 ID05-1030004000, KJC005 Lung Tissue C TGC0094 C.posadasii 3656 lung tissue TGC0095 C. posadasii 4038 ID05- 10500004038,KJD003 Chin Abscess C TGC0096 C. posadasii 7835 ID05- 2310007835, KJA010Bronchial wash C TGC0097 C. posadasii 4273 ID05- 1150004273, KJC006 LungTissue C TGC0098 C. posadasii 4581 ID05- 1240004581, KJB005c Bronchialwash C TGC0099 C. posadasii 4644 ID05- 1260004644, KJK002a Systemic;urogenital C TGC0100 C. posadasii 4645 body fluid TGC0102 C. posadasii4947 ID05-1360004947, KJA005 Sputum C TGC0103 C. posadasii 4948ID05-1360004948, KJA006 Human C TGC0104 C. posadasii 5003 ID05-1370005003, KJM002 Sputum C TGC0105 C. posadasii 5032 ID05-1380005032Human C TGC0106 C. posadasii 5354 ID05- 1470005354, KJF003 Sputum CTGC0107 Unknown 11227 bronch wash TGC0108 C. posadasii 11276 scalp massTGC0109 C. posadasii 11309 bronch wash TGC0110 Unknown 11311 bronch washTGC0111 C. posadasii 11318 sputum TGC0112 C. posadasii 11337thorocentesis TGC0113 C. posadasii 11436 RUL fluid TGC0114 C. posadasii11461 bronch wash TGC0115 C. posadasii 11467 leukens trap (?) TGC0116 C.posadasii 11482 sputum TGC0117 Unknown 11524 BAL TGC0118 C. posadasii11491 back wound TGC0119 C. posadasii 11610 bronch wash TGC0120 C.posadasii 11615 sputum TGC0121 C. posadasii 11625 BAL RML TGC0122 C.posadasii 11676 lymph node TGC0123 C. posadasii 11723 bronch washTGC0124 C. posadasii B16  BT05- 0810000016, KJG002 Pulmonary C TGC0125C. posadasii BT17 BT05- 0810000017, KJG003 Wound C TGC0126 C. posadasiiBT18 BT05- 0810000018, KJG004 Wound C TGC0127 C. posadasii BT19 BT05-0810000019, KJG005 Pulmonary C TGC0128 Unknown 378 lung tissue TGC0129Unknown 559 unknown TGC0130 C. posadasii 632 unknown TGC0131 C.posadasii 633 unknown TGC0132 C. posadasii 713 sputum TGC0133 C.posadasii 745 unknown TGC0134 C. posadasii 1155 BAL TGC0135 C. immitis2010 Human C TGC0136 C. immitis 2012 RMSCC 2012 Human C TGC0137 C.posadasii 3131 RMSCC 1038, f0634 Lung C TGC0138 C. posadasii 1042 RMSCC1042 ~ ~ TGC0139 C. immitis 2017 RMSCC 2017 Human C TGC0140 C. posadasii3142 RMSCC 1049, f0925 Cerebral spinal fluid C TGC0141 C. immitis 2271RMSCC 2271 Human C TGC0142 C. posadasii 3137 RMSCC 1054 Urine, CSF CTGC0143 C. immitis 3505 RMSCC 3505 Human C TGC0144 C. posadasii 3101RMSCC 1439, f1053 Sputum C TGC0145 C. posadasii 113 lymph node TGC0146Unknown 561 unknown TGC0147 C. posadasii 114 RL Bx TGC0148 C. posadasii211 sputum TGC0149 C. posadasii 223 RUL mass TGC0150 C. posadasii 660 LLmass TGC0151 C. posadasii 5783 lung mass TGC0152 C. posadasii 3570bronch wash TGC0153 C. posadasii 6452 ID05- 1860006452, KJH004d Sputum CTGC0154 C. posadasii 6453 ID05- 1860006453, KJH005d Bronchial wash CTGC0155 C. posadasii 6472 ID05- 1870006472, KJA009 Bronchial wash CTGC0156 C. posadasii 6826 ID05- 1990006826, KJE003 Bronchial wash CTGC0157 C. posadasii 6831 ID05- 1990006831, KJD004 Testicular Abscess CTGC0158 C. posadasii 6994 ID05- 2030006994, KJB008 Pleural fluid CTGC0159 C. posadasii 7471 ID05-2200007471 Lung tissue C TGC0160 C.posadasii 7527 ID05- 2210007527, KJC009 Pulmonary C TGC0173 C. posadasiiID05-10154 TGC0174 Unknown PT523004/ PT323004 TGC0175 C. posadasiiPT318013  TGC0176 C. posadasii PT07291021 TGC0177 C. posadasiiPT07290023 TGC0178 C. posadasii PT320028  TGC0179 C. posadasii PT299001 TGC0180 Unknown PT07288010 TGC0181 C. posadasii PT299006  TGC0182Unknown PT305012  TGC0183 C. posadasii PT305013  TGC0184 UnknownPT07292005 TGC0185 Unknown PT312004  TGC0186 Unknown PT296022  TGC0187Unknown PT07292004 TGC0188 Unknown PT07250001 TGC0189 C. posadasiiPT07269001 TGC0190 C. posadasii PT07270023 TGC0191 C. posadasiiPT07276015 TGC0192 Unknown PT298017/ PT268017 TGC0193 UnknownPT07250037/ PT07250032 TGC0194 Unknown PT324006  TGC0195 C. posadasiiPT296001  TGC0196 C. posadasii PT07254016 TGC0197 C. posadasiiPT07268016 TGC0198 Unknown PT07260017 TGC0199 C. posadasii PT07291022TGC0200 Unknown PT07290022 TGC0201 C. posadasii PT295029  TGC0202 C.posadasii PT268018  TGC0203 C. posadasii PT309005  TGC0204 UnknownPT305014  TGC0205 C. posadasii PT07278010 TGC0206 Unknown PT305010 TGC0207 Unknown PT309003  TGC0208 C. posadasii PT303019  TGC0209 C.posadasii PT305011  TGC0210 C. posadasii PT306003  TGC0211 C. posadasii3196 f1202 Shoulder C TGC0212 C. posadasii 3215 f1132 Skin biopsy CTGC0213 C. immitis 3377 RMSCC 3377 Human C TGC0214 C. posadasii VFC040F0777 Lung tissue C TGC0216 C. immitis 3476 TGC0217 C. posadasii 3351f0675 Blood C TGC0218 C. posadasii 3177 f1281 Bronchoalveolar C lavageTGC0219 C. posadasii 3251 f0961 Lung C TGC0220 C. posadasii 3322 f1002Elbow abcess fluid C TGC0221 C. posadasii 3315 f0940 Lung C TGC0222 C.posadasii 3232 f1244 Sputum C TGC0223 C. posadasii VFC013 FF512 Sputum CTGC0224 C. posadasii 3474 f0106 Lung lesion C TGC0226 C. posadasii 3250f0960 Blood C TGC0227 C. posadasii 3312 RMSCC 3312 ~ ~ TGC0228 C.posadasii 3492 f0813 Bronch trans C TGC0229 C. posadasii 3311 RMSCC 3311~ ~ TGC0230 C. posadasii 3278 f1070 Sputum C TGC0231 C. posadasii 3317f0944 Blood C TGC0232 C. posadasii 3248 f0953 TGH mold iso C TGC0233 C.posadasii 3247 Human C TGC0234 C. posadasii 3268 f1037 Blood C TGC0235C. posadasii 3262 f1020 Bronchoalveolar C lavage TGC0236 C. posadasii3275 f1066 Lung C TGC0237 C. posadasii 3234 f1258 Bronchoalveolar Clavage TGC0238 C. posadasii 3319 f0994 Bronchoalveolar C lavage TGC0239C. posadasii 3240 f1265 Sputum C TGC0240 C. posadasii 3299 f0875 SputumC TGC0241 C. posadasii 3256 f0982 Bronchoalveolar C lavage TGC0242 C.posadasii 3236 Human C TGC0243 C. posadasii 3272 f1049 Lung C TGC0244 C.posadasii 3273 f1062 Sputum C TGC0245 C. posadasii 3239 f1264 Sputum CTGC0246 C. posadasii 3318 f0988 Human C TGC0247 C. posadasii 3286TGC0248 C. posadasii 3230 f1242 Sputum C TGC0249 C. posadasii 1030000010 Urine C TGC0250 C. posadasii 3231 Human C TGC0251 C. posadasii3237 f1262 Sputum C TGC0252 C. posadasii 3305 f0903 Lung aspirate CTGC0253 C. posadasii 3289 f0830 Sputum C TGC0254 C. posadasii 3221 HumanC TGC0255 C. posadasii 3294 TGC0256 C. posadasii 3284 f0754 Sputum CTGC0257 C. posadasii 3223 Human C TGC0258 C. immitis 2281 RMSCC 2281 ~ ~TGC0259 C. posadasii 3300 f0876 Lung aspirate C TGC0260 C. posadasii3482 f0163 Brain C TGC0261 C. posadasii 3238 f1263 Pleural fluid CTGC0262 C. posadasii 3263 f1021 Lung mold bronch C TGC0263 C. posadasii3224 f1090 Bronchial wash C TGC0264 C. posadasii 3253 f0969 Lung noduleC TGC0265 C. posadasii 3211 f1142 Blood C TGC0266 C. posadasii 3285TGC0267 C. posadasii 3214 f1134 Lymph node C TGC0268 C. posadasii 3308f0915 Human C TGC0269 C. posadasii 3252 f0964 TGH C TGC0270 C. posadasii3293 f0848 Tracheal aspirate C TGC0271 C. posadasii 3245 f1274Bronchoalveolar C lavage TGC0272 C. posadasii 3213 f1141 Sputum CTGC0273 C. posadasii 3226 Human C TGC0274 C. posadasii 3298 f0874 SputumC TGC0275 C. posadasii 3219 f1116 Bronchial wash C TGC0276 C. posadasii3216 f1130 Bronchial C TGC0277 C. posadasii 3246 f1275 Human C TGC0278C. posadasii 3295 Human C TGC0279 C. posadasii 3227 f1239 Sputum CTGC0280 C. posadasii 3277 Human C TGC0281 C. posadasii 3217 f1124 SputumC TGC0282 C. posadasii 3269 f1040 Arm C TGC0283 C. posadasii 3279 f1071Sputum C TGC0284 C. posadasii 3169 f1294 Sputum C TGC0285 C. posadasiiVFC014 FF532 Lymph node C TGC0286 C. posadasii 3165 f1306 Face lesion CTGC0287 C. posadasii 3192 f1215 Sputum C TGC0288 C. posadasii 3205 f1170Bronchoalveolar C lavage TGC0289 C. posadasii 3207 f1159 Lung aspirate CTGC0290 C. posadasii VFC010 FF180 Sputum C TGC0291 C. posadasii VFC007FF877 Human C TGC0292 C. posadasii 3356 f0691 Bronchial wash C TGC0293C. immitis 2267 RMSCC 2267 ~ ~ [VFC 052 2267] TGC0294 C. posadasiiVFC008 FF861 Sputum C TGC0295 C. posadasii VFC005 FF795 Sputum C TGC0296C. posadasii VFC024 FF625 Forearm fluid C TGC0297 C. posadasii VFC022FF690 Sputum C TGC0298 C. posadasii 3132 f1038 Bronchoalveolar C lavageTGC0299 C. posadasii VFC019 FF616 Human C TGC0300 C. posadasii 3199f1193 Pleural tissue C TGC0301 C. posadasii 3209 f1153 Pleural fluid CTGC0302 C. posadasii 3203 Human C TGC0303 C. posadasii 3357 f0697 SputumC TGC0304 C. posadasii 3167 f1285 Sputum C TGC0305 C. posadasii 3162f1300 Knee fluid C TGC0306 C. posadasii 3202 f1185 Bronchus C TGC0307 C.posadasii VFC006 FF826 Bronchial wash C TGC0308 C. posadasii 3121 f0987Lung Aspirate C TGC0309 C. immitis 3706 RMSCC 3706 Human C TGC0310 C.posadasii VFC036 FF538 Lung C TGC0311 C. posadasii VFC047 97-300-0319Cerebral spinal fluid C TGC0312 C. posadasii VFC009 FF83 Sputum CTGC0313 C. posadasii 3183 f1234 Lung C TGC0314 C. posadasii VFC029 FF167Abdominal wall C tissue TGC0315 C. immitis 3475 RMSCC 3475 ~ ~ TGC0316C. posadasii 3190 f1219 Blood C TGC0317 C. posadasii VFC011 FF379 Lungaspirate C TGC0318 C. posadasii 3208 f1158 Sputum C TGC0319 C. immitis2008 RMSCC 2008, VFC043 Human C [VFC 043 2008] TGC0320 C. posadasiiVFC030 FF340 FNA lung C TGC0321 C. immitis 2395 RMSCC 2395 Human CTGC0322 C. posadasii VFC012 FF449 Lung C TGC0323 C. posadasii VFC033FF387 Sputum C TGC0324 C. posadasii VFC032 FF368 Sputum C TGC0325 C.posadasii 3355 f0688 Bronchial wash C TGC0326 C. posadasii VFC021/VFC02CAPD fluid C 18 TGC0327 C. posadasii 3163 f1302 Sputum C TGC0328 C.posadasii 3346 f0656 Lung tissue C TGC0329 C. posadasii 3333 f0562 Neckdrainage C TGC0330 C. posadasii VFC037 FF612 Sputum C TGC0331 C.posadasii 3193 f1210 Blood C TGC0332 C. posadasii VFC034 FF455 Sputum CTGC0333 C. posadasii VFC031 FF361 Sputum C TGC0334 C. posadasii VFC028FF170 Sputum C TGC0335 C. posadasii VFC017 FF551 Sputum C TGC0336 C.posadasii 3174 f1277 Sputum C TGC0337 C. posadasii VFC016 FF555Bronchial aveolar C lavage TGC0338 C. posadasii VFC025 FF715 Sputum CTGC0339 C. posadasii 3187 f1223 Urine C TGC0340 C. posadasii 3491 f1257Human C TGC0341 C. posadasii 3457 f0193 Skin C TGC0342 C. immitis 2279RMSCC 2279 ~ ~ TGC0343 C. immitis 2268 RMSCC 2268 ~ ~ TGC0344 C.posadasii 184 ID02-184 Bronchial wash C TGC0345 C. posadasii 3409 f0263Sputum C TGC0346 C. posadasii VFC020 FF641 Lung aspirate C TGC0347 C.posadasii VFC002 FF416 Human C TGC0348 C. posadasii 3175 f1278 Human CTGC0349 C. posadasii 3395 f0388 Blood C TGC0350 C. posadasii 587id03-587 Bronchial wash C TGC0351 C. immitis 2269 RMSCC 2269 ~ ~ TGC0352C. posadasii 3198 f1194 Bronchoalveolar C lavage TGC0353 C. posadasiiVFC003 FF430 Bronchial tissue C TGC0354 C. posadasii 3172 f1287 Lungaspirate C TGC0355 C. posadasii 3398 f0391 Bronchial wash C TGC0356 C.posadasii CPA0085 a03-6397 Cat C TGC0357 C. posadasii Mar-78 TGC0358 C.posadasii 3160 f1298 Femur C TGC0359 C. posadasii 3206 f1164 Human CTGC0360 C. posadasii 3335 f0565 Sputum C TGC0361 C. posadasii 3413 f0269Human C TGC0362 C. posadasii 3538 0204-3538 Pleural fluid C TGC0363 C.posadasii 5786 0204-5786 Bone marrow C TGC0364 C. posadasii 3336 f0577Sputum C TGC0365 C. posadasii 3168 f1295 Bronchoalveolar C lavageTGC0366 C. posadasii 3186 f1224 Bronchoalveolar C lavage TGC0367 C.posadasii 517 id03-517 Bronchial wash C TGC0368 C. posadasii 584id03-584 Sputum C TGC0370 C. posadasii VFC018 FF584 Sputum C TGC0371 C.immitis 3693 RMSCC 3693 ~ ~ TGC0372 C. posadasii 3180 f1266 Human CTGC0373 C. posadasii 5127 0205-5127 Bronchial wash C TGC0374 C. immitis2278 RMSCC 2278 ~ ~ TGC0375 C. posadasii 3489 f1222 Human C TGC0376 C.posadasii 3128 RMSCC 1036, B3128, f0415, Wound C [VFC 049] VFC049TGC0379 C. posadasii 3411 f0267 Bronchus C TGC0380 C. posadasii CPA0088a03-8172 Cat C TGC0381 C. immitis 2277 RMSCC 2277 Human C TGC0382 C.posadasii 3201 f1190 Bronchoalveolar C lavage TGC0383 C. posadasii 2294TGC0384 C. posadasii 3166 f1309 Bronchoalveolar C lavage TGC0385 C.posadasii 4165 0204-4165 Sputum C TGC0386 C. posadasii 9888 0204-9888Bronchial wash C TGC0387 C. posadasii 7892 0204-7892 Sputum C TGC0388 C.posadasii 3157 RMSCC 1040 Bronchoalveolar C lavage TGC0389 C. posadasii3488 RMSCC 3488 Human C TGC0390 C. posadasii C735 Lab Strain, Patient Cisolation TGC0391 C. posadasii Silveira Lab Strain, Patient C isolationTGC0392 C. posadasii CPA0066 604-1 L Soil E TGC0393 C. immitis RSVaccine/Lab Strain, C Patient isolation TGC0394 C. posadasii 3310 f0920Sputum C TGC0395 C. posadasii 3314 f0939 TTA C TGC0396 C. posadasii 3145RMSCC 1037 Skin biopsy C TGC0398 C. posadasii 2133 RMSCC 2133 Human CTGC0399 C. posadasii CPA0020 485B-0 L Soil E TGC0400 C. immitis 2394RMSCC 2394 Human C TGC0401 C. posadasii CPA0001 407-0 L Soil E TGC0402C. immitis 3703 RMSCC 3703 Human C TGC0403 C. immitis 2014 RMSCC 2014 ~~ TGC0405 C. posadasii 3290 f0931 Sputum C TGC0406 C. posadasii 3352f0676 Sputum C TGC0408 C. posadasii 3301 f0877 Bronchoalveolar C lavageTGC0409 C. posadasii 3220 f1107 Lung biopsy C TGC0410 C. posadasii 3244f1273 Bronchoalveolar C lavage TGC0413 C. immitis 2009 RMSCC 2009 ~ ~TGC0414 C. posadasii 3283 f1087 Sputum C TGC0415 C. posadasii 3243 f1270Bone marrow C TGC0416 C. immitis 2274 RMSCC 2274 ~ ~ TGC0417 C.posadasii 3276 f1067 Bronchoalveolar C lavage TGC0418 C. posadasii 3222Human C TGC0419 C. posadasii 3321 Human C TGC0420 C. posadasii 3233f1247 Pleural fluid C TGC0421 C. posadasii 3228 f1240 Lung C TGC0422 C.posadasii 3291 f0832 Sputum C TGC0423 C. posadasii 3229 f1241 Tissuemass C TGC0424 C. posadasii 3292 f0843 Sputum C TGC0425 C. posadasii3354 f0687 Skin biopsy C TGC0426 C. immitis 2280 RMSCC 2280 ~ ~ TGC0427C. posadasii 3326 f0537 Lung nodule C TGC0428 C. posadasii 3296 Human CTGC0429 C. immitis 2015 RMSCC 2015 ~ ~ TGC0430 C. immitis 2273 RMSCC2273 ~ ~ TGC0431 C. posadasii 3249 f0956 Bronchus C TGC0432 C. posadasii3313 f0936 SE C TGC0434 C. immitis 2011 RMSCC 2011 ~ ~ TGC0435 C.posadasii 3353 f0678 Bronchial wash C TGC0436 C. posadasii 3344 f0650Bronchial wash C TGC0437 C. posadasii 3184 f1233 Thoracentesis C TGC0438C. posadasii 3210 f1143 Bronchoalveolar C lavage TGC0439 C. posadasiiVFC038 FF569 Pleural fluid C TGC0440 C. posadasii 3161 f1299 Lung CTGC0441 C. posadasii 3185 f1232 Lung aspirate C TGC0442 C. posadasii3197 f1196 Lung biopsy C TGC0443 C. posadasii 3181 f1236 Sputum CTGC0444 C. posadasii 2372 TGC0445 C. posadasii 3191 f1217 Lung biopsy CTGC0446 C. posadasii VFC039 FF670 Sputum C TGC0447 C. immitis 3705 RMSCC3705 ~ ~ TGC0448 C. posadasii 3375 f0339 Lung biopsy C TGC0449 C.posadasii 3348 f0664 Lung aspirate C TGC0450 C. posadasii 3178 f1282Bronchoalveolar C lavage TGC0451 C. posadasii 3350 Human C TGC0452 C.posadasii 3188 f1221 Sputum C TGC0453 C. posadasii 3347 f0658 Transbronch biopsy C TGC0455 C. immitis 2105 RMSCC 2105, VFC050 Human CTGC0456 C. immitis 2006 RMSCC 2006 ~ ~ TGC0457 C. posadasii 3349 Human CTGC0458 C. posadasii 3171 f1289 Sputum C TGC0459 C. posadasii VFC057KUM4825 Abdominal wall C tissue TGC0460 C. posadasii 3139 f1010 Sputum CTGC0461 C. posadasii 3170 f1293 Sputum C TGC0462 C. posadasii CPA0087a03-7777 Dog C TGC0463 C. posadasii 2127 RMSCC 2127, VFC041 Human CTGC0464 C. posadasii 3179 f1283 Joint fluid C TGC0465 C. immitis 2102RMSCC 2102 ~ ~ TGC0466 C. posadasii VFC001 F415 Forehead lesion Caspirate TGC0467 C. posadasii 3164 f1305 Sputum C TGC0469 C. posadasii3136 f1133 Sputum C TGC0470 C. posadasii 3200 f1192 Urine C TGC0471 C.immitis 2275 RMSCC 2275 Human C TGC0472 C. posadasii 8959 0204-8959Bronchial wash C TGC0473 C. immitis 2276 RMSCC 2276 ~ ~ TGC0474 C.posadasii 59 ID02-59 Lung biopsy C TGC0475 C. posadasii 3490 f1256 LungC TGC0476 C. posadasii 927 id03-927 Lung biopsy C TGC0477 C. immitis3479 RMSCC 3479 Human C TGC0478 C. posadasii 1D02-60 TGC0480 C.posadasii 3374 f0337 Skin biopsy C TGC0481 C. posadasii 3338 0205-3338Bronchial wash C TGC0482 C. posadasii 3381 f0353 Sputum C TGC0483 C.posadasii 3368 f0328 Lung C TGC0484 C. posadasii 3129 0205-3129 Wound CTGC0485 C. posadasii 4885 0205-4885 Bronchial wash C TGC0486 C.posadasii 3420 f0280 Pleural fluid C TGC0487 C. posadasii Mar-87 TGC0488C. posadasii 3440 f0322 Human C TGC0489 C. posadasii CPA0086 a03-7379Llama C TGC0490 C. posadasii 674 id03-674 Lung nodule C TGC0492 C.posadasii 49 ID02-49 Bone marrow C TGC0493 C. posadasii 699 id03-699Bronchoalveolar C lavage TGC0494 C. posadasii 3364 f0738 Shoulder CTGC0495 C. posadasii 179 ID03-179 Sputum C TGC0496 C. posadasii 81570204-8157 Bronchial wash C TGC0497 C. posadasii 7874/0204-7074 0204-7874Bronchial wash C TGC0498 C. posadasii 9968 0203-9968 Bronchial wash CTGC0499 C. posadasii CPA0083 a03-4364 Cat C TGC0500 C. posadasii 81740204-8174 Chest fluid C TGC0501 C. posadasii 3391 TGC0502 C. posadasii3417 f0276 Bronchial wash C TGC0503 C. posadasii 147 ID02-147 Bronchialwash C TGC0504 C. posadasii 3447 f0079 Urine C TGC0505 C. posadasiih20289 Human C TGC0506 C. posadasii 8911 0205-8911 Blood C TGC0507 C.posadasii 263 ID02-263 Sputum C TGC0508 C. posadasii 3439 f0321 Human CTGC0509 C. posadasii 3360 f0718 Sputum C TGC0511 C. posadasii 3341 f0591Sputum C TGC0512 C. posadasii 3345 f0652 Bone marrow C TGC0513 C.posadasii 7076 0204-7076 Lung tissue C TGC0514 C. posadasii 3358 f0700Bronchial wash C TGC0515 C. immitis 3382 f0354 Gastric aspirate CTGC0517 C. posadasii PT08141001 Forehead Wound C TGC0518 C. posadasiiPT08140024 Bronch Wash C TGC0519 Unknown PT08164001 Bronch Wash CTGC0520 C. posadasii PT08148001 Bronch Wash C TGC0521 Unknown PT08172013Bronch Wash C TGC0522 C. posadasii PT08140028 BAL C TGC0523 C. posadasiiPT08128012 Bronch Wash C TGC0524 C. posadasii PT08129008 CSF C TGC0525C. posadasii PT08126016 Sputum C TGC0526 C. posadasii PT08137008 SputumC TGC0527 C. posadasii PT08084019 Pleural Fluid C TGC0528 C. posadasiiPT08091007 Bronch Wash C TGC0529 Unknown PT08115001 Bronch Wash CTGC0530 Unknown PT08103001 Sputum C TGC0531 Unknown PT08099015 BronchWash C TGC0532 Unknown PT08100013 R. Chest tissue C TGC0533 UnknownPT08189001 Bronch Wash C TGC0534 Unknown PT08196009 Hip Joint C TGC0535Unknown PT08192009 Sputum C TGC0536 Unknown PT08200005 R. Upper Lung CTGC0537 Unknown PT08172014 Bronch Wash C TGC0538 Unknown PT08189002Bronch Wash C TGC0539 Unknown PT08179006 Left lung C TGC0540 UnknownPT08171012 BAL C TGC0541 Unknown PT08224005 Rigth lung mass C TGC0542Unknown PT08205002 Bronch Wash C TGC0543 Unknown PT08206002 Bronch SwabC TGC0544 Unknown PT08200006 Bronch Wash C TGC0545 Unknown PT08224008Bronch C TGC0546 Unknown PT08217008 Bronch Wash C TGC0547 UnknownPT08226004 Unknown C TGC0548 Unknown PT08203016 Sputum C TGC0552 UnknownPT08240019 Sputum TGC0553 Unknown PT08228008 Abscess, back TGC0554Unknown PT08235003 Sputum TGC0555 Unknown PT08219008 Sputum TGC0556Unknown PT08228009 Leg Wound TGC0557 Unknown PT08256022 Bronch WashTGC0558 Unknown PT08231010 Lymph Node TGC0559 Unknown PT08197007 BALTGC0560 Unknown PT08256023 Sputum TGC0561 Unknown PT08169002 KneeTGC0562 Unknown PT08266012 Right Lower Lung TGC0563 Unknown PT08267009BAL TGC0564 Unknown PT08246015 CSF TGC0565 Unknown TG06812 SputumTGC0566 Unknown TG6480 Sputum TGC0567 Unknown PT08288016 TGC0568 UnknownPT08297002 TGC0569 Unknown PT08297001 TGC0570 Unknown PT08294003 TGC0571Unknown PT08301009 TGC0572 Unknown PT08302004 TGC0573 Unknown PT08301010TGC0574 Unknown PT08303030 TGC0575 Unknown PT08288012 TGC0576 UnknownPT08303031 TGC0577 Unknown PT08284003 TGC0578 Unknown PT08283001 TGC0579Unknown PT08288010 TGC0580 Unknown PT08319002 TGC0581 Unknown PT08304014TGC0582 Unknown PT08288009 TGC0583 Unknown PT08246023 TGC0584 UnknownPT08308015 TGC0585 Unknown PT08283003 TGC0586 Unknown PT08246025 TGC0587Unknown PT08246024 TGC0588 Unknown PT08246016 TGC0589 C. immitisCDCtransplant1 C TGC0590 C. immitis CDCtransplant2 C TGC0591 C. immitisCDCtransplant3 C TGC0648 C. posadasii 2343 RMSCC 2343 ~ ~ TGC0649 C.posadasii 2346 RMSCC 2346 ~ ~ TGC0650 C. posadasii 3472 RMSCC 3472 ~ ~TGC0651 C. posadasii 3480 RMSCC 3480 ~ ~ TGC0652 C. posadasii 3487 RMSCC3487 ~ ~ TGC0655 C. posadasii 3506 RMSCC 3506 ~ ~

Additionally, a direct comparison of CocciEnv and CocciDxQ showed thatthe CocciEnv assay results in an average of 1.81 and range of 1.56 to2.05 in Ct values earlier than that from the CocciDxQ assay whenscreened on the same DNA (Table 11). This translates to an almost 4-foldhigher capture of Coccidioides DNA in a sample.

TABLE 11 Coccidioides genomic DNA screened for a side-by-side comparisonof the CocciDxQ and CocciEnv assays. CocciEnv CocciDxQ Ct valuedifference Sample qPCR qPCR between CocciEnv (in duplicate) Ct value Ctvalue and CocciDxQ TGC0004_1-3 21.18 22.92 1.74 TGC0004_1-3 21.09 22.931.85 TGC0213_1-3 21.37 23.14 1.77 TGC0213_1-3 21.32 23.12 1.80TGC0222_1-3 21.17 23.00 1.83 TGC0222_1-3 21.22 23.05 1.83 TGC0293_1-319.24 21.11 1.88 TGC0293_1-3 19.28 21.20 1.92 TGC0306_1-3 19.38 21.271.89 TGC0306_1-3 19.30 21.24 1.95 TGC0319_1-3 16.75 18.66 1.90TGC0319_1-3 16.80 18.50 1.70 TGC0332_1-3 17.68 19.66 1.98 TGC0332_1-317.67 19.64 1.97 TGC0350_1-3 18.57 20.57 2.00 TGC0350_1-3 18.49 20.542.05 TGC0385_1-3 17.31 19.30 2.00 TGC0385_1-3 17.25 19.28 2.03TGC0392_1-3 26.49 28.46 1.97 TGC0392_1-3 26.49 28.43 1.94 TGC0396_1-319.00 20.98 1.97 TGC0396_1-3 18.85 20.91 2.06 TGC0404_1-3 14.60 16.561.96 TGC0404_1-3 14.68 16.50 1.82 TGC0408_1-3 18.90 20.61 1.71TGC0408_1-3 18.85 20.60 1.75 TGC0434_1-3 16.97 18.58 1.61 TGC0434_1-316.99 18.55 1.56 TGC0442_1-3 20.58 22.21 1.63 TGC0442_1-3 20.62 22.201.58 TGC0450_1-3 19.79 21.50 1.70 TGC0450_1-3 19.81 21.53 1.72TGC0453_1-3 17.98 19.81 1.83 TGC0453_1-3 17.91 19.81 1.90 TGC0458_1-319.98 21.58 1.61 TGC0458_1-3 19.81 21.57 1.76 TGC0467_1-3 20.09 21.821.73 TGC0467_1-3 20.02 21.81 1.79 TGC0470_1-3 18.90 20.68 1.78TGC0470_1-3 18.91 20.55 1.64 TGC0473_1-3 20.74 22.42 1.67 TGC0473_1-320.77 22.35 1.58 TGC0478_1-3 17.64 19.25 1.61 TGC0478_1-3 17.65 19.301.65 TGC0528_1-3 35.12 36.79 1.66 TGC0528_1-3 Negative 36.91 Average no.cycles of earlier amplification 1.81 of gDNA with CocciEnv versusCocciDxQ assay

We claim:
 1. A method of determining the presence or absence ofCoccidioides in a DNA-containing sample comprising the steps of: addinga first and a second oligonucleotide capable of binding SEQ ID NO. 1 toa mixture comprising the DNA-containing sample, wherein the firstoligonucleotide includes at least one sequence selected from the groupconsisting of SEQ ID NOs: 10-20 and oligonucleotides having at least 90%sequence identity to any one of SEQ ID NOs: 10-20, wherein the secondoligonucleotide includes at least one sequence selected from the groupcomprising SEQ ID NOs: 21-38, and oligonucleotides having at least 90%sequence identity to any one of SEQ ID NOs: 21-38; subjecting themixture containing the first and second oligonucleotides to conditionsthat allow amplification of nucleic acid comprising the firstoligonucleotide; obtaining a result indicating nucleic acidamplification comprising the first oligonucleotide; and determining thepresence or absence of Coccidioides in the DNA-containing sample basedon the result.
 2. The method of claim 1, wherein the result comprises aCt value.
 3. The method of claim 1, wherein the first oligonucleotide iscapable of hybridizing with complements of SEQ ID NO: 10, and the secondoligonucleotide is capable of hybridizing with complements of SEQ ID NO:21 in the mixture.
 4. The method of claim 1, further comprising the stepof adding a third oligonucleotide to the mixture, wherein the thirdoligonucleotide binds to its complement included in the amplificationproducts by the first and second oligonucleotides.
 5. The method ofclaim 4, wherein the third oligonucleotide includes a sequence selectedfrom the group consisting of SEQ ID NO. 2 and oligonucleotides having atleast 90% sequence identity to SEQ ID NO.
 2. 6. The method of claim 4,wherein at least one of the first, the second and the thirdoligonucleotides comprises a label.
 7. The method of claim 6, whereinthe label comprises a fluorescent label.
 8. The methods of claim 6,wherein the third oligonucleotide comprises a fluorescent label.
 9. Themethod of claim 1, further comprising the step of isolating DNA from theDNA-containing sample.
 10. The method of claim 1, wherein the samplecomprises an environmental sample.
 11. The method of claim 10, whereinthe environmental sample comprises a soil sample.
 12. The method ofclaim 1, wherein the sample is derived from a subject.
 13. The method ofclaim 12, wherein the subject is selected from the group consisting of ahuman, a companion animal, a domesticated animal, a livestock animal,and a wild animal species.
 14. A method of quantifying Coccidioides in aDNA-containing sample comprising the steps of: adding a first and asecond oligonucleotide capable of binding SEQ ID NO. 1 to a mixturecomprising the DNA-containing sample, wherein the first oligonucleotideincludes at least one sequence selected from the group consisting of SEQID NOs: 10-20 and oligonucleotides having at least 90% sequence identityto any one of SEQ ID NOs: 10-20, wherein the second oligonucleotideincludes at least one sequence selected from the group comprising SEQ IDNOs: 21-38, and oligonucleotides having at least 90% sequence identityto any one of SEQ ID NOs: 21-38; subjecting the mixture containing thefirst and second oligonucleotides to conditions that allow amplificationof a template DNA comprising the first oligonucleotide; obtaining afirst result indicating amplification of the template DNA andCoccidioides quantification; and calculating Coccidioides quantificationbased on the first result in comparison to a reference result, whereinCoccidioides quantification determines the amount of template DNA in thesample.
 15. The method of claim 14, wherein the reference result isobtained by amplification of a DNA-containing sample having a knownquantity of Coccidioides.
 16. The method of claim 14, wherein thereference result is predetermined.
 17. The method of claim 14, whereinthe first and the reference result each comprises a Ct value.
 18. Themethod of claim 14, further comprising the step of adding a thirdoligonucleotide to the mixture, wherein the third oligonucleotide bindsto its complement included in the amplification products by the firstand second oligonucleotides.
 19. The method of claim 18, wherein thethird oligonucleotide includes a sequence selected from the groupconsisting of SEQ ID NO. 2 and oligonucleotides having at least 90%sequence identity to SEQ ID NO:
 2. 20. The method of claim 18, whereinat least one of the first, the second, and the third oligonucleotidescomprises a label.
 21. The method of claim 20, wherein the labelcomprises a fluorescent label.
 22. The method of claim 20, wherein thethird oligonucleotide comprises a fluorescent label.
 23. The method ofclaim 14, further comprising the step of isolating DNA from theDNA-containing sample.
 24. The method of claim 14, wherein the samplecomprises an environmental sample.
 25. The method of claim 24, whereinthe environmental sample comprises a soil sample.
 26. The method ofclaim 14, wherein the sample is derived from a subject, and furtherwherein the subject is selected from the group consisting of a human, acompanion animal, a domesticated animal, a livestock animal, and a wildanimal species.